WO2018152687A1 - Anti-lymphocyte activation gene-3 (lag-3) antibodies and uses thereof - Google Patents

Anti-lymphocyte activation gene-3 (lag-3) antibodies and uses thereof Download PDF

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Publication number
WO2018152687A1
WO2018152687A1 PCT/CN2017/074365 CN2017074365W WO2018152687A1 WO 2018152687 A1 WO2018152687 A1 WO 2018152687A1 CN 2017074365 W CN2017074365 W CN 2017074365W WO 2018152687 A1 WO2018152687 A1 WO 2018152687A1
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Prior art keywords
seq
amino acid
acid sequence
chain variable
variable domain
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PCT/CN2017/074365
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French (fr)
Inventor
Lei Fang
Zhengyi WANG
Bingshi GUO
Jingwu Zang
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I-Mab
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Priority to PCT/CN2017/074365 priority Critical patent/WO2018152687A1/en
Priority to CA3053989A priority patent/CA3053989A1/en
Priority to KR1020187022954A priority patent/KR102144317B1/en
Priority to KR1020197014275A priority patent/KR102236259B1/en
Priority to EP18733152.5A priority patent/EP3389702A4/en
Priority to PCT/CN2018/076940 priority patent/WO2018153340A1/en
Priority to JP2019544658A priority patent/JP6929951B2/en
Priority to US16/069,487 priority patent/US10577421B2/en
Priority to SG11201907561PA priority patent/SG11201907561PA/en
Priority to AU2018226298A priority patent/AU2018226298B2/en
Priority to NZ756678A priority patent/NZ756678A/en
Priority to CN201880000932.2A priority patent/CN109475617B/en
Publication of WO2018152687A1 publication Critical patent/WO2018152687A1/en
Priority to IL26873419A priority patent/IL268734A/en
Priority to US16/687,387 priority patent/US11414485B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates generally to the field of molecular biology and protein biochemistry. More specifically, the invention relates to antibodies that bind to Lymphocyte Activation Gene-3 (LAG-3) and methods of use thereof.
  • LAG-3 Lymphocyte Activation Gene-3
  • Lymphocyte Activation Gene-3 (LAG-3) (also known as CD223) is a member of the immunoglobulin (Ig) superfamily, is closely related to CD4, and variously impacts T cell function. LAG-3 is expressed on activated T cells, exhausted T cells, tumor infiltrating T cells, and regulatory T cells (T regs ) . Upon binding with major histocompatibility complex 2 (MHC class II) , the LAG-3/MHC class II interaction results in the negative regulation of T cell proliferation, activation, and homeostasis.
  • MHC class II major histocompatibility complex 2
  • LAG-3 represents an important immune checkpoint in cancer, similarly to cytotoxic T lymphocyte antigen-4 (CTLA-4) , programmed cell death ligand-1 (PD-L1) , and programmed cell death-1 (PD-1) .
  • CTLA-4 cytotoxic T lymphocyte antigen-4
  • PD-L1 programmed cell death ligand-1
  • PD-1 programmed cell death-1
  • LAG-3 not only expresses on the activated/exhausted effector T cells but also on regulatory T cells. LAG3 antagonism can not only promote the activation of effector T cells, but also block the suppressive function of regulatory T cells. Therefore, LAG-3 represents a promising target for cancer immunotherapy and preclinical evidence suggests that an anti-LAG-3 antibody can promote an anti-tumor response.
  • anti-LAG-3 mAbs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following:
  • MHC major histocompatibility complex
  • the antibodies of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with LAG-3 in humans and animals, including the prevention and treatment of solid and hematological cancers.
  • the antibodies of the disclosure are also useful as diagnostics to determine the level of LAG-3 expression in tissue samples.
  • Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof; pharmaceutical compositions comprising one or more of the anti-LAG-3 monoclonal antibodies, preferably chimeric or humanized forms of said antibodies; methods of therapeutic use of such anti-LAG-3 monoclonal antibodies; and cell lines that produce these anti-LAG-3 monoclonal antibodies.
  • the embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined by reference to specific structural characteristics i.e. specified amino acid sequences of either the CDRs or entire heavy chain or light chain variable domains. All of these antibodies bind to LAG-3.
  • the monoclonal antibodies, or antigen binding fragments thereof may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide.
  • an antibody comprises at least one light chain comprising the three light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy chain comprising the three heavy chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.
  • Preferred embodiments are anti-LAG-3 mAbs, or antigen binding fragments thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2; said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 4; and said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:
  • the heavy chain variable domain may comprise any one of the listed variable heavy chain CDR1 sequences (HCDR1) in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR3 sequences (HCDR3) .
  • HCDR1 and HCDR2 and HCDR3 are particularly preferred, which derive from a single common V H domain, examples of which are described herein.
  • the antibody or antigen binding fragment thereof may additionally comprise a light chain variable domain (V L ) , which is paired with the V H domain to form an antigen binding domain.
  • V L light chain variable domain
  • Preferred light chain variable domains are those comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, S
  • the light chain variable domain may comprise any one of the listed variable light chain CDR1 sequences (LCDR1) in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR3 sequences (LCDR3) .
  • LCDR1 and LCDR2 and LCDR3 are particularly preferred, which derive from a single common V L domain, examples of which are described herein.
  • any given LAG-3 antibody or antigen binding fragment thereof comprising a V H domain paired with a V L domain will comprise a combination of 6 CDRs: variable heavy chain CDR1 (HCDR1) , variable heavy chain CDR2 (HCDR2) , variable heavy chain CDR3 (HCDR3) , variable light chain CDR1 (LCDR1) , variable light chain CDR2 (LCDR2) , and variable light chain CDR1 (LCDR1) .
  • HCDR1 variable heavy chain CDR1
  • HCDR2 variable heavy chain CDR2
  • HCDR3 variable heavy chain CDR3
  • LCDR1 variable light chain CDR1
  • LCDR1 variable light chain CDR1
  • LCDR1 variable light chain CDR1
  • Preferred combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDR1 (HCDR1) , variable heavy chai n CDR2 (HCDR2) , variable heavy chain CDR3 (HCDR3) , variable light chain CDR1 (LCDR1) , variable light chain CDR2 (LCDR2) , and variable light chain CDR3 (LCDR3) selected from the group consisting of:
  • HCDR1 comprising SEQ ID NO: 1
  • HCDR2 comprising SEQ ID NO: 3
  • HCDR3 comprising SEQ ID NO: 5
  • LCDR1 comprising SEQ ID NO: 46
  • LCDR2 comprising SEQ ID NO: 81
  • LCDR3 comprising SEQ ID NO: 104;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 6, LCDR1 comprising SEQ ID NO: 47, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 7, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 8, LCDR1 comprising SEQ ID NO: 49, LCDR2 comprising SEQ ID NO: 83, LCDR3 comprising SEQ ID NO: 105;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 9, LCDR1 comprising SEQ ID NO: 50, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106;
  • HCDR1 comprising SEQ ID NO: 1
  • HCDR2 comprising SEQ ID NO: 3
  • HCDR3 comprising SEQ ID NO: 10
  • LCDR1 comprising SEQ ID NO: 48
  • LCDR2 comprising SEQ ID NO: 82
  • LCDR3 comprising SEQ ID NO: 107;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 11, LCDR1 comprising SEQ ID NO: 51, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 108;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 109;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 13, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 104;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 14, LCDR1 comprising SEQ ID NO: 53, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 110;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 16, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 17, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 18, LCDR1 comprising SEQ ID NO: 56, LCDR2 comprising SEQ ID NO: 87, LCDR3 comprising SEQ ID NO: 113;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 19, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 20, LCDR1 comprising SEQ ID NO: 57, LCDR2 comprising SEQ ID NO: 98, LCDR3 comprising SEQ ID NO: 114;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 115;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 22, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 116;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 23, LCDR1 comprising SEQ ID NO: 59, LCDR2 comprising SEQ ID NO: 88, LCDR3 comprising SEQ ID NO: 117;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 24, LCDR1 comprising SEQ ID NO: 60, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 25, LCDR1 comprising SEQ ID NO: 61, LCDR2 comprising SEQ ID NO: 89, LCDR3 comprising SEQ ID NO: 118;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 27, LCDR1 comprising SEQ ID NO: 63, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 111;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 28, LCDR1 comprising SEQ ID NO: 65, LCDR2 comprising SEQ ID NO: 91, LCDR3 comprising SEQ ID NO: 120;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 29, LCDR1 comprising SEQ ID NO: 66, LCDR2 comprising SEQ ID NO: 92, LCDR3 comprising SEQ ID NO: 121;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 30, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 93, LCDR3 comprising SEQ ID NO: 122;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 67, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 123;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 32, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 124;
  • HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 33, LCDR1 comprising SEQ ID NO: 68, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 125;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 34
  • LCDR1 comprising SEQ ID NO: 69
  • LCDR2 comprising SEQ ID NO: 85
  • LCDR3 comprising SEQ ID NO: 126;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 35
  • LCDR1 comprising SEQ ID NO: 70
  • LCDR2 comprising SEQ ID NO: 94
  • LCDR3 comprising SEQ ID NO: 127;
  • HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 128;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 36
  • LCDR1 comprising SEQ ID NO: 71
  • LCDR2 comprising SEQ ID NO: 86
  • LCDR3 comprising SEQ ID NO: 129;
  • HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 115, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 130;
  • HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 34, LCDR1 comprising SEQ ID NO: 70, LCDR2 comprising SEQ ID NO: 96, LCDR3 comprising SEQ ID NO: 131;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 37
  • LCDR1 comprising SEQ ID NO: 72
  • LCDR2 comprising SEQ ID NO: 97
  • LCDR3 comprising SEQ ID NO: 132;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 38
  • LCDR1 comprising SEQ ID NO: 72
  • LCDR2 comprising SEQ ID NO: 97
  • LCDR3 comprising SEQ ID NO: 132;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 39
  • LCDR1 comprising SEQ ID NO: 73
  • LCDR2 comprising SEQ ID NO: 86
  • LCDR3 comprising SEQ ID NO: 133;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 40
  • LCDR1 comprising SEQ ID NO: 74
  • LCDR2 comprising SEQ ID NO: 99
  • LCDR3 comprising SEQ ID NO: 134;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 41
  • LCDR1 comprising SEQ ID NO: 75
  • LCDR2 comprising SEQ ID NO: 93
  • LCDR3 comprising SEQ ID NO: 135;
  • HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 76, LCDR2 comprising SEQ ID NO: 100, LCDR3 comprising SEQ ID NO: 130;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 42
  • LCDR1 comprising SEQ ID NO: 76
  • LCDR2 comprising SEQ ID NO: 100
  • LCDR3 comprising SEQ ID NO: 136;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 43
  • LCDR1 comprising SEQ ID NO: 77
  • LCDR2 comprising SEQ ID NO: 101
  • LCDR3 comprising SEQ ID NO: 137;
  • HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 35, LCDR1 comprising SEQ ID NO: 78, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 138;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 44
  • LCDR1 comprising SEQ ID NO: 79
  • LCDR2 comprising SEQ ID NO: 102
  • LCDR3 comprising SEQ ID NO: 122;
  • HCDR1 comprising SEQ ID NO: 2
  • HCDR2 comprising SEQ ID NO: 4
  • HCDR3 comprising SEQ ID NO: 45
  • LCDR1 comprising SEQ ID NO: 80
  • LCDR2 comprising SEQ ID NO: 103
  • LCDR3 comprising SEQ ID NO: 139.
  • anti-LAG-3 antibodies include antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain (V H ) having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO
  • preferred anti-LAG-3 antibodies including antibodies or antigen binding fragments thereof may comprise a light chain variable domain (V L ) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
  • preferred LAG-3 antibodies, or antigen binding fragments thereof are those comprising a combination of a heavy chain variable domain (V H ) and a light chain variable domain (V L ) , wherein the combination is selected from the group consisting of:
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 140 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 189;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 141 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 190;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 142 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 191;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 143 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 192;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 194;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 195;
  • viii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 147 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 196;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 148 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 197;
  • x a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 149 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 198;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 199;
  • xii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 151 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 200;
  • xiii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 201;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 153 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 202;
  • xv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 203;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 204;
  • xvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 205;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 206;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 207;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 208;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 209;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 210;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 211;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 212;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 213;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 165 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 214;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 215;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 167 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 216;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 217;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 218;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 219;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 220;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 221;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 173 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 222;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 174 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 223;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 175 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 224;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 176 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 225;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 226;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 227;
  • xl a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 179 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 228;
  • xli a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 180 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 229;
  • xlii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 181 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 230;
  • xliii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 182 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 231;
  • xliv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 183 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 232;
  • xlv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 184 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 233;
  • a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 185 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 234;
  • xlvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 186 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 235;
  • xlviii a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 236;
  • xlix a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 237.
  • Preferred anti-LAG-3 antibodies or antigen binding fragments thereof may also comprise a combination of a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a V H sequence with at least 85%sequence identity, or at least 90%sequence identity, or at least 95%sequence identity, or at least 97%, 98%or 99%sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xlix) and/or the light chain variable domain comprises a V L sequence with at least 85%sequence identity, or at least 90%sequence identity, or at least 95%sequence identity, or at least 97%, 98%or 99%sequence identity, to the light chain amino acid sequences shown above in (i) to (xlix) .
  • the specific VH and V L pairings or combinations in parts (i) through (xlix) may be preserved for anti-LAG-3 antibodies having V H and V L domain sequences with a particular percentage sequence identity to these reference sequences
  • the V H and/or V L domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
  • the preferred anti-LAG-3 antibodies described herein are also characterized by combinations of properties which are not exhibited by prior art anti-LAG-3 antibodies proposed for human therapeutic use. Accordingly, the preferred anti-LAG-3 antibodies described herein are characterized by:
  • MHC major histocompatibility complex
  • the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat LAG-3.
  • the anti-LAG-3 mAbs disclosed are contemplated herein.
  • the anti-LAG-3 mAbs can be full length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgG1, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchangeor an antibody fragment, e.g., a F (ab') 2 fragment, a F (ab) fragment, a single chain Fv fragment (scFv) , etc., as disclosed herein.
  • an antibody fragment e.g., a F (ab') 2 fragment, a F (ab) fragment, a single chain Fv fragment (scFv) , etc., as disclosed herein.
  • compositions comprising one or more of the anti-LAG-3 mAbs or fragments disclosed herein, optionally chimeric or humanized forms, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • anti-LAG-3 mAbs that possess the functional profiles as described herein.
  • the anti-LAG-3 mAbs of the present disclosure exhibit distinct combinations of properties, particularly combinations of properties that render the mAbs particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers, ischemia-reperfusion injury, autoimmune and/or inflammatory diseases.
  • FIG. 1 The D1-D2 domains are critical for LAG-3 function. Wildtype (WT) LAG3 extracellular domain (ECD) fusion protein (LAG-3-ECD-huFc) fragments can bind to Daudi cells while D1-D2 truncated LAG-3-ECD-huFc fragments fail to bind Daudi cells.
  • WT Wildtype
  • ECD extracellular domain
  • LAG-3-ECD-huFc LAG-3-ECD-huFc
  • FIG. 2 The binding of human anti-LAG3 antibodies to LAG3 protein derived from various species. Anti-LAG-3 antibodies were evaluated for their binding properties to human, rat, and mouse LAG3 through enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 3 The binding of human anti-LAG3 antibodies to cell surface LAG-3 antigen on activated human primary CD4 + T cells.
  • Anti-LAG-3 antibodies were assessed for binding to cell surface LAG-3 antigen on activated human primary CD4 + T cells at various concentrations (10 ⁇ g/ml, 3.333 ⁇ g/ml, 1.111 ⁇ g/ml, 0.370 ⁇ g/ml, 0.123 ⁇ g/ml, 0.041 ⁇ g/ml, 0.014 ⁇ g/ml and 0.005 ⁇ g/ml) .
  • FIG. 4 Inhibition of soluble LAG-3 (sLAG) binding to MHC class II receptor by anti-LAG-3 antibody.
  • Anti-LAG-3 antibodies were evaluated for their ability to block the binding of sLAG-3 to MHC class II receptor in an in vitro binding assay using biotin-labeled LAG-3-ECD-huFcLAG-3-Fc fusion proteins and Raji cells expressing MHC class II receptor.
  • FIG. 5 Stimulation of IL-2 production in peripheral blood mononuclear cells (PBMCs) by anti-LAG-3 antibodies.
  • Anti-LAG-3 antibodies were administrated into Staphylococcal Enterotoxin B (SEB) stimulated PBMC at various concentrations starting from 20 ⁇ g/ml at 1: 3 serious dilution for 6 doses.
  • SEB Staphylococcal Enterotoxin B
  • IL-2 concentration in the culture supernatant was evaluated by enzyme-linked immunosorbent assay (ELISA) .
  • FIG. 6 Reversing the suppressive function of regulatory T cells (T regs ) on effector T cells (T effs ) using anti-LAG-3 antibodies.
  • T regs regulatory T cells
  • T effs effector T cells
  • FIG. 7 Synergistic effect of anti-LAG3 and PD-1 antibody combo treatment.
  • the anti-LAG3 antibodies were tested in combination with PD-1 antibody on SEB-stimulated PBMCs assay.
  • FIG. 8 Anti-LAG-3 antibodies enhance human T cell response in the presence of PD-L1 antibody.
  • the anti-. antibodies were evaluated in combination with PD-L1 antibody on human mixed lymphocyte reaction (MLR) assay.
  • MLR human mixed lymphocyte reaction
  • the present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, which bind to human LAG-3 and that have desirable functional properties.
  • the antibodies of the invention are derived from particular heavy and light chain germline sequences and/or comprise particular structural features such as CDR regions comprising particular amino acid sequences.
  • This disclosure provides isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies and pharmaceutical compositions containing the antibodies, immunoconjugates or bispecific molecules of the invention.
  • This disclosure also relates to methods of using the antibodies, such as to detect LAG-3 protein, as well as to methods of using the anti-LAG-3 antibodies of the invention to stimulate immune responses, alone or in combination with other immunostimulatory antibodies. Accordingly, this disclosure also provides methods of using the anti-LAG-3 antibodies of the invention to, for example, inhibit tumor growth or treat viral infection.
  • LAG-3 refers to Lymphocyte Activation Gene-3.
  • the LAG3 protein which belongs to immunoglobulin (Ig) superfamily, comprises a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains, designated D1 to D4.
  • Ig immunoglobulin
  • the term “LAG-3” includes variants, isoforms, homologs, orthologs, and paralogs.
  • antibodies specific for a human LAG-3 protein may, in certain cases, cross-react with a LAG-3 protein from a species other than human.
  • the antibodies specific for a human LAG-3 protein may be completely specific for the human LAG-3 protein and may not exhibit species or other types of cross-reactivity, or may cross-react with LAG-3 from certain other species but not all other species (e.g., cross-react with monkey LAG-3, but not mouse LAG-3) .
  • human LAG-3 refers to human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 having GenBank Accession No. NP 002277.
  • mouse LAG-3 refers to mouse sequence LAG-3, such as the complete amino acid sequence of mouse LAG-3 having GenBank Accession No. NP 032505.
  • LAG-3 is also known in the art as, for example, CD223.
  • the human LAG-3 sequence may differ from human LAG-3 of GenBank Accession No. NP 002277 by having, e.g., conserved mutations or mutations in non-conserved regions and the LAG-3 has substantially the same biological function as the human LAG-3 of GenBank Accession No. NP 002277.
  • a biological function of human LAG-3 is having an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody of the instant disclosure or a biological function of human LAG-3 is binding to MHC Class II molecules.
  • a particular human LAG-3 sequence will generally be at least 90%identical in amino acids sequence to human LAG-3 of GenBank Accession No. NP 002277 and contains amino acid residues that identify the amino acid sequence as being human when compared to LAG-3 amino acid sequences of other species (e.g., murine) .
  • a human LAG-3 can be at least 95%, or even at least 96%, 97%, 98%, or 99%identical in amino acid sequence to LAG-3 of GenBank Accession No. NP 002277.
  • a human LAG-3 sequence will display no more than 10 amino acid differences from the LAG-3 sequence of GenBank Accession No. NP 002277.
  • the human LAG-3 can display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the LAG-3 sequence of GenBank Accession No. NP 002277. Percent identity can be determined as described herein.
  • immune response refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • an "antigen-specific T cell response” refers to responses by a T cell that result from stimulation of the T cell with the antigen for which the T cell is specific.
  • responses by a T cell upon antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production) .
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion” ) or single chains thereof.
  • Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H 1, C H 2, and C H 3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDRs complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a LAG-3 protein) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H 1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab'fragment, which is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3. sup. rd ed.
  • the two domains of the F v fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain F v (scFv) ; see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (I988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) .
  • Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody.
  • These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • an "isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a LAG-3 protein is substantially free of antibodies that specifically bind antigens other than LAG-3 proteins) .
  • An isolated antibody that specifically binds a human LAG-3 protein may, however, have cross reactivity to other antigens, such as LAG-3 proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • the term "human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans-chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below) , (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • a host cell transformed to express the human antibody e.g., from a transfectoma
  • c antibodies isolated from a recombinant, combinatorial human antibody library
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • isotype refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen. "
  • human antibody derivatives refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • an antibody that "specifically binds human LAG-3" is intended to refer to an antibody that binds to human LAG-3 protein (and possibly a LAG-3 protein from one or more non-human species) but does not substantially bind to non-LAG-3 proteins.
  • the antibody binds to a human LAG-3 protein with "high affinity” , namely with a K D of 1 ⁇ 10 -7 M or less, more preferably 5 ⁇ 10 -8 M or less, more preferably 3 ⁇ 10 -8 M or less, more preferably 1 ⁇ 10 -8 M or less, more preferably 25 ⁇ 10 -9 M or less or even more preferably 1 ⁇ 10 -9 M or less.
  • does not substantially bind to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K D of 1 ⁇ 10 -6 M or more, more preferably 1 ⁇ 10 -5 M or more, more preferably 1 ⁇ 10 -4 M or more, more preferably 1 ⁇ 10 -3 M or more, even more preferably 1 ⁇ 10 -2 M or more.
  • K assoc or "K a " , as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction
  • K dis or "K d , " as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K d /K a ) and is expressed as a molar concentration (M) .
  • K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a system.
  • high affinity for an IgG antibody refers to an antibody having a K D of 1 x 10 -7 M or less, more preferably 5 x 10 -8 M or less, even more preferably 1 x 10 -8 M or less, even more preferably 5 x 10 -9 M or less, and even more preferably 1 x 10 -9 M or less for a target antigen.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to an antibody having a K D of 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less.
  • subject includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
  • the antibodies of the invention are characterized by particular functional features or properties of the antibodies.
  • the antibodies specifically bind to human LAG-3 and may bind to LAG-3 from certain other species, e.g., monkey LAG-3, e.g., cynomolgus monkey, rhesus monkey, but may not substantially bind to LAG-3 from certain other species, e.g., mouse LAG-3.
  • an antibody of the invention binds to human LAG-3 with high affinity.
  • an antibody of the invention binds to human LAG-3 and exhibits an ability to stimulate an antigen-specific T cell response. In other embodiments, an antibody of the invention binds to human LAG-3 but does not exhibit an ability to stimulate an antigen-specific T cell response.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon gamma
  • Other means by which to evaluate the ability of the antibody to stimulate an immune response include the ability of the antibody to inhibit tumor growth, such as in an in vivo tumor graft model or the ability of the antibody to stimulate an autoimmune response, such as the ability to promote the development of an autoimmune disease in an autoimmune model, such as the ability to promote the development of diabetes in the NOD mouse model.
  • an antibody of the invention can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human LAG-3, such as CHO cells that have been transfected to express LAG-3, e.g., human LAG-3, or monkey LAG-3, e.g., rhesus or cynomolgus monkey or mouse LAG-3 on their cell surface.
  • a cell line that expresses human LAG-3 such as CHO cells that have been transfected to express LAG-3, e.g., human LAG-3, or monkey LAG-3, e.g., rhesus or cynomolgus monkey or mouse LAG-3 on their cell surface.
  • suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4 + activated T cells, which express native LAG-3.
  • the binding of the antibody can be tested in BIAcore binding assays.
  • suitable binding assays include ELISA assays, for example using a recombinant LAG-3 protein.
  • an antibody of the invention binds to a LAG-3 protein with a K D of 5 x 10 -8 M or less, binds to a LAG-3 protein with a K D of 2 x 10 -8 M or less, binds to a LAG-3 protein with a K D of 5 x 10 -9 M or less, binds to a LAG-3 protein with a K D of 4 x 10 -9 M or less, binds to a LAG-3 protein with a K D of 3 x 10 -9 M or less, binds to a LAG-3 protein with a K D of 2 x 10 -9 M or less, binds to a LAG-3 protein with a K D of 125 x 10 -9 M or less, binds to a LAG-3 protein with a K D of 5 x 10 -10 M or less, or binds to a LAG-3 protein with a K D of 1 x 10 -10 M or less.
  • Preferred antibodies of the invention are the human monoclonal antibodies S27, S31, T99, and S119 isolated and structurally characterized as described [Examples 2-8] .
  • the V H amino acid sequences of S27, S31, T99 and S119 are shown in SEQ ID NO: 149, SEQ NO: 150, SEQ ID NO: 158, and SEQ ID NO: 162, respectively.
  • the V L amino acid sequences of S27, S31, T99, and S119 are shown in SEQ ID NO: 198, SEQ NO: 199, SEQ ID NO: 207, and SEQ ID NO: 211, respectively.
  • V H and V L sequences can be "mixed and matched" to create other anti-LAG-3 binding molecules of the invention.
  • V H and V L chains are mixed and matched, a V H sequence from a particular V H /V L pairing is replaced with a structurally similar V H sequence.
  • a V L sequence from a particular V H /V L pairing is replaced with a structurally similar V L sequence.
  • this disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof comprising:
  • the antibody specifically binds human LAG-3.
  • variable heavy and variable light chain combinations include:
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 199;
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 207;
  • a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 211.
  • the CDR3 domain independently from the CDR1and/or CDR2 domain (s) , alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., Brit. J. of Can. 83 (2) : 252-60, 2000; Beiboer et al., J. Mol. Biol.
  • the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from an antibody derived from a human or non-human animal, wherein the monoclonal antibody is capable of specifically binding to human LAG-3.
  • the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to LAG-3.
  • inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody.
  • the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a human antibody, such as, e.g., a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to human LAG-3.
  • the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to human LAG-3 and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for LAG-3 to generate a second human antibody that is capable of specifically binding to human LAG-3.
  • a first human antibody such as, for example, a human antibody obtained from a non-human animal
  • the first human antibody is capable of specifically binding to human LAG-3
  • the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for LAG-3 to generate a second human antibody that is capable of specifically binding to human LAG-3.
  • inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental first human antibody.
  • an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-LAG-3 antibodies of the invention.
  • this disclosure provides an isolated monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 140-188; (b) the light chain variable region comprises an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 189-237; (c) the antibody specifically binds to human LAG-3; (d) blocks LAG-3 binding to major histocompatibility complex (MHC) class II molecules; (e) stimulates an immune response; and (f) reverses the inhibitory effect of regulatory T cells on effector cells.
  • MHC major histocompatibility complex
  • the antibody can possess one or more of the following functional properties discussed above, such as high affinity binding to human LAG-3, binding to monkey LAG-3, lack of binding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHC Class II molecules and/or the ability to stimulate antigen-specific T cell responses.
  • the antibody can be, for example, a human antibody, a humanized antibody or a chimeric antibody.
  • the V H and/or V L amino acid sequences can be 85%, 90%, 95%, 96%, 97%, 98%, or 99%homologous to the sequences set forth above.
  • An antibody having V H and V L regions having high (i.e., 80%or greater) homology to the V H and V L regions of the sequences set forth above can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acids of V H and/or V L amino acid sequences, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth above) using the functional assays described herein.
  • mutagenesis e.g., site-directed or PCR-mediated mutagenesis
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4: 11-7, 1988) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-53, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against public databases to, e.g., to identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (J. Mol. Biol. 215: 403-10, 1990) .
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucl. Acid Res. 25 (17) : 3389-402, 1997) .
  • the default parameters of the respective programs e.g., XBLAST and NBLAST are useful.
  • the terms “humanized” , “humanization” , and the like refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (Methods, 36 (1) : 25-34, 2005) and Hou et al. (J. Biochem. 144 (1) : 115-20, 2008) , respectively, to improve various antibody properties, as discussed below.
  • FR or "framework sequence” refers to any one of FRs 1 to 4.
  • Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present. For example, this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human.
  • Substantially human frameworks are those that have at least 80%sequence identity to a known human germline framework sequence.
  • the substantially human frameworks have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.
  • Fully human frameworks are those that are identical to a known human germline framework sequence.
  • Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.
  • IMGT ImMunoGeneTics
  • the Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences.
  • the individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci.
  • germline light chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39 and IGKV1D-33 and IGKJ1-5 and germline heavy chain FRs can be selected from the group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20
  • Substantially human FRs are those that have at least 80%sequence identity to a known human germline FR sequence.
  • the substantially human frameworks have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to a CDR sequence disclosed herein.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein.
  • Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.
  • Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods Almagro et al. (Front. Biosci., Humanization of antibodies Jan 1 (13) : 1619-33, 2008) .
  • the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework.
  • the sequence identity of the new framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identical to the sequence of the corresponding framework in the parent antibody compound.
  • frameworks having fewer than 100 amino acid residues one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody.
  • the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (PNAS 88: 2869, 1991) .
  • Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in vicieri et al. (Bioinformatics Feb 1; 31 (3) : 434-5, 2015) and U.S. Patent Nos.
  • the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework.
  • the sequence identity of the new FR will generally be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the corresponding FR in the parent antibody compound.
  • FRs having fewer than 100 amino acid residues one, two, three, four, five, or more amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody.
  • the FR can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in vicieri et al. Bioinformatics. 2015 Feb 1; 31 (3) : 434-435 and U.S. Patents 4,816,397, 5,225,539, and 5,693,761; and the method of Winter and co-workers (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536.
  • the amino acid in the human FR of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position;
  • any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three-dimensional immunoglobulin model.
  • Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds.
  • Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties.
  • Single mutations producing improved properties can be combined to assess their effects in combination with one another.
  • amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein.
  • amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.
  • murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (V L ) and variable heavy (V H ) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site.
  • CDRs complementarity determining regions
  • V L variable light
  • V H variable heavy
  • the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients.
  • SDR grafting a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody-ligand interaction, called “SDR grafting” , wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks.
  • SDR grafting involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen–antibody complexes of known structures, or by mutational analysis of the antibody-combining site.
  • Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated mAbs can contain the set of CDRs from a single murine mAb disclosed herein, or light and heavy chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs.
  • Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a “mix and match” approach to create novel mAbs comprising a mixture of CDRs from the disclosed murine mAbs to achieve new, or improved, therapeutic activities.
  • Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that "compete" with the molecules disclosed herein are those that bind human LAG-3 at site (s) that are identical to, or overlapping with, the site (s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human LAG-3 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labeled.
  • the labeled compound and the unlabeled compound bind to separate and discrete sites on LAG-3, the labeled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind.
  • competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to LAG-3 by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
  • Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Such assays can be made quantitative by using purified antibodies.
  • a standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor.
  • the capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.
  • mAbs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods disclosed in the Examples presented below.
  • competing antibodies possess about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below.
  • antibodies of the invention can be engineered to include modifications within the Fc region of any of the isotypes described, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity (ADCC) .
  • an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • ADCC antigen-dependent cellular cytotoxicity
  • an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the mAbs or antigen-binding fragments thereof, or competing antibodies useful in the compositions and methods can be any of the isotypes described herein.
  • any of these isotypes can comprise further amino acid modifications as follows.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG1 isotype.
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life.
  • Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007) .
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al., 2006, Yeung et al., 2009) ; M252Y/S254T/T256E (Dall’A cqua et al., 2006) ; T250Q/M428L (Hinton et al., 2006) ; and M428L/N434S (Zalevsky et al., 2010) .
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to amino acid modifications I253A (Petkova et al., 2006) ; P257I/N434H, D376V/N434H (Datta-Mannan et al., 2007) ; and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005) .
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) , Antibody-Dependent Cellular Phagocytosis (ADCP) , C1q binding, and altered binding to Fc receptors.
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al., 2001) ; S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006) ; F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al., 2007) ; G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008) ; K326A/E333A, K326A/E333S and
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al., 1993, Walker et al., 1989) ; L234A/L235A (Xu et al., 2000) ; K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al., 2008) ; C226S/C229S/E233P/L234V/L235A (McEarchern et al., 2007) ; S267E/L328F (Chu et al., 2008) .
  • the human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999) ; E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) , Antibody-Dependent Cellular Phagocytosis (ADCP) , and C1q binding, and altered binding to Fc receptors.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to the amino acid modification K326A/E333S (Idusogie et al., 2001) .
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999) ; E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S
  • the Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (C H1 domain) , C232S, C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightle et al., 2010) .
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.
  • the human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid (s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) , or apoptosis activity.
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • apoptosis activity apoptosis activity.
  • the human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP) .
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to amino acid modifications F234A/L235A (Alegre et al., 1994) ; S228P, L235E and S228P/L235E (Reddy et al., 2000) .
  • the glycosylation of an antibody is modified.
  • a glycosylated antibody can be made (i.e., the antibody lacks glycosylation) .
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such a glycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • An antibody can be PEGylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • PEG polyethylene glycol
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the PEGylation is carried out via an acylation reaction or an alkylation reaction with are active PEG molecule (or an analogous reactive water-soluble polymer) .
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy-oraryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0154316 and EP 0401384.
  • Antibodies of the invention can be tested for binding to human LAG-3 by, for example, standard ELISA.
  • Anti-LAG-3 human IgG antibodies can be further tested for reactivity with a LAG-3 antigen by Western blotting.
  • the binding specificity of an antibody of the invention can also be determined by monitoring binding of the antibody to cells expressing a LAG-3 protein, e.g., flow cytometry. These methods are known in the art. See, e.g., Harlow and Lane (1988) , cited supra.
  • Antibodies of this invention can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC) .
  • Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents.
  • the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker.
  • the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.
  • the ADC scan be prepared as described in U.S. Patent Nos.
  • bispecific molecules comprising an anti-LAG-3 antibody linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • a bispecific molecule includes molecules that have three or more specificities.
  • the bispecific molecule comprises a first binding specificity for LAG-3 and a second binding specificity for a triggering molecule that recruits cytotoxic effector cells that can kill a LAG-3 expressing target cell.
  • suitable triggering molecules are CD64, CD89, CD16, and CD3. See, e.g., Kufer et al., Trends in Biotech. 22 (5) : 238-44, 2004.
  • a bispecific molecule has, in addition to an anti-Fc binding specificity and an anti-LAG-3 binding specificity, a third specificity.
  • the third specificity can be for an anti-enhancement factor (EF) , e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell.
  • EF anti-enhancement factor
  • the anti-enhancement factor can bind a cytotoxic T cell (e.g. via CD2, CD3, CDS, CD28, CD4, CD40, or ICAM-1) , other immune regulatory molecules (e.g.
  • PD-1 via PD-1, PD-L1, CTLA-4, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, CD47 or CD73) or other immune cell, resulting in an increased immune response against the target cell.
  • Bispecific molecules can come in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's ) linked by a peptide chain, a so-called Bs (scFv) 2 construct. Intermediate-sized bispecific molecules include two different F (ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the present disclosure formulated together with a pharmaceutically acceptable earlier. It may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug.
  • the pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immunostimulatory agent, anti-cancer agent, an anti-viral agent, or a vaccine, such that the anti-LAG-3 antibody enhances the immune response against the vaccine.
  • the pharmaceutical composition can comprise any number of excipients.
  • Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003) , the disclosure of which is incorporated herein by reference.
  • a pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active compound can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
  • an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
  • the pharmaceutical compounds of the invention can be in the form of pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01%to about ninety-nine percent of active ingredient, preferably from about 0.1%to about 70%, most preferably from about 1%to about 30%of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dosage ranges from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every 3 to 6 months.
  • Preferred dosage regimens for an anti-LAG-3antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g/mL and in some methods about 25-300 ⁇ g/mL.
  • a “therapeutically effective dosage” of an anti-LAG-3 antibody of the invention preferably results in a decrease in severity of disease symptoms, an increase infrequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a "therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%relative to untreated subjects.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
  • the pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556) ; (2) micro-infusion pumps (U.S. Patent No. 4,487,603) ; (3) transdermal devices (U.S. Patent No. 4,486,194) ; (4) infusion apparati (U.S. Patent Nos. 4,447,233 and 4,447,224) ; and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196) ; the disclosures of which are incorporated herein by reference.
  • needleless hypodermic injection devices e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,
  • the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo.
  • the therapeutic compounds of the invention can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade, J. Clin. Pharmacol. 29: 685, 1989; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al. (1995) FEBSLett.
  • the antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, detection of LAG-3 or enhancement of immune response by blockade of LAG-3.
  • the antibodies of the present invention are human antibodies.
  • these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations.
  • the invention provides a method of modifying an immune response in a subject comprising administering to the subject the antibody, or antigen-binding portion thereof, of the invention such that the immune response in the subject is modified.
  • the response is enhanced, stimulated or up-regulated.
  • Preferred subjects include human patients in need of enhancement of an immune response.
  • the methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., the T-cell mediated immune response) .
  • the methods are particularly suitable for treatment of cancer in vivo.
  • the anti-LAG-3 antibodies can be administered together with an antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor bearing or virus-bearing subject) .
  • the two can be administered in either order or simultaneously.
  • the invention further provides methods for detecting the presence of humanLAG-3 antigen in a sample, or measuring the amount of human LAG-3 antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to human LAG-3, under conditions that allow for formation of a complex between the antibody or portion thereof and human LAG-3. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of human LAG-3 antigen in the sample.
  • the anti-LAG-3 antibodies of the invention can be used to purify human LAG-3 via immunoaffinity purification.
  • the invention also provides in vitro and in vivo methods of using the antibodies of the invention to stimulate, enhance or upregulate antigen-specific T cell responses.
  • the invention provides a method of stimulating an antigen-specific T cell response comprising contacting said T cell with the antibody of the invention such that an antigen-specific T cell response is stimulated.
  • Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response.
  • suitable indicators include increased T cell proliferation in the presence of the antibody and/or increase cytokine production in the presence of the antibody.
  • interleukin-2 production by the antigen specific T cell is stimulated.
  • the invention also provides a method of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject comprising administering an antibody of the invention to the subject such that an immune response (e.g., an antigen-specific T cell response) in the subject is stimulated.
  • an immune response e.g., an antigen-specific T cell response
  • the subject is a tumor-bearing subject and an immune response against the tumor is stimulated.
  • the subject is a virus-bearing subject and an immune response against the virus is stimulated.
  • the invention provides a method for inhibiting growth of tumor cells in a subject comprising administering to the subject an antibody of the invention such that growth of the tumor is inhibited in the subject.
  • the invention provides a method of treating viral infection in a subject comprising administering to the subject an antibody of the invention such that the viral infection is treated in the subject.
  • Blockade of LAG-3 by antibodies can enhance the immune response to cancerous cells in the patient.
  • the present invention relates to treatment of a subject in vivo using an anti-LAG-3 antibody such that growth of cancerous tumors is inhibited.
  • An anti-LAG-3 antibody can be used alone to inhibit the growth of cancerous tumors.
  • an anti-LAG-3 antibody can be used in conjunction with other immunogenic agents, standard cancer treatments, or other antibodies, as described below.
  • the invention provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-LAG-3 antibody, or antigen-binding portion thereof.
  • the antibody is a human anti-LAG-3 antibody (such as any of the human anti-human LAG-3 antibodies described herein) .
  • the antibody can be a chimeric or humanized anti-LAG-3 antibody.
  • Preferred cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy.
  • preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma) , renal cancer (e.g. clear cell carcinoma) , prostate cancer (e.g., hormone refractory prostate adenocarcinoma) , breast cancer, colon cancer and lung cancer (e.g., non-small cell lung cancer) .
  • the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention.
  • cancers examples include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood,
  • antibodies to LAG-3 can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , cells, and cells transfected with genes encoding immune stimulating cytokines (He et al (2004) J. Immunol. 173: 4919-28) .
  • tumor vaccines include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below) .
  • tumors have been shown to be immunogenic such as melanomas.
  • LAG-3 blockade By raising the threshold of T cell activation by LAG-3 blockade, the tumor responses in the host can be activated.
  • LAG-3 blockade is likely to be more effective when combined with a vaccination protocol.
  • Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds. ) , 1997, Cancer: Principles and Practice of Oncology, Fifth Edition) .
  • a vaccine is prepare dusing autologous or allogeneic tumor cells.
  • These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF.
  • GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S. A. 90: 3539-43) .
  • tumor specific antigens are differentiation antigens expressed in the tumors and in the cell from which the tumor arose, for example melanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be the targets of tumor specific T cells found in the host. LAG-3 blockade can be used in conjunction with a collection of recombinant proteins and/or peptides expressed in a tumor in order to generate an immune response to these proteins.
  • the tumor antigen can include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85%of human cancers and in only a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013) . (These somatic tissues may be protected from immune attack by various means) .
  • Tumor antigen can also be "neo-antigens" expressed in cancer cells because of somatic mutations that alter protein sequence or create fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome) , or idiotype from B cell tumors.
  • tumor vaccines can include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV) , Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV) .
  • HPV Human Papilloma Viruses
  • HBV and HCV Hepatitis Viruses
  • KHSV Kaposi's Herpes Sarcoma Virus
  • Another form of tumor specific antigen which can be used in conjunction with LAG-3 blockade is purified heat shock proteins (HSP) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from the tumor cells and these HSPs are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot &Srivastava (1995) Science269: 1585-1588; Tamura et al. (1997) Science 278: 117-120) .
  • DC Dendritic cells
  • DC's can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332) .
  • DCs can also be transduced by genetic means to express these tumor antigens as well.
  • DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al. (2000) Nature Medicine 6: 332-336) .
  • As a method of vaccination DC immunization can be effectively combined with LAG-3 blockade to activate more potent anti-tumor responses.
  • LAG-3 blockade can also be combined with standard cancer treatments. LAG-3 blockade can be effectively combined with chemotherapeutic regimes. In these instances, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304) .
  • An example of such a combination is an anti-LAG-3 antibody in combination with decarbazine for the treatment of melanoma.
  • Another example of such a combination is an anti-LAG-3antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma.
  • IL-2 interleukin-2
  • LAG-3 blockade The scientific rationale behind the combined use of LAG-3 blockade and chemotherapy is that cell death, that is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
  • Other combination therapies that may result in synergy with LAG-3 blockade through cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.
  • Angiogenesis inhibitors can also be combined with LAG-3 blockade. Inhibition of angiogenesis leads to tumor cell death which may feed tumor antigen into host antigen presentation pathways.
  • LAG-3 blocking antibodies can also be used in combination with bispecific antibodies that target Fca or Fey receptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243) .
  • Bispecific antibodies can be used totarget two separate antigens.
  • anti-Fc receptor/anti-tumor antigen e.g., Her-2/neu
  • bispecific antibodies have been used to target macrophages to sites of tumor. This targeting may more effectively activate tumor specific responses.
  • the T cell arm of these responses would be augmented by the use of LAG-3 blockade.
  • antigen may be delivered directly to DCs by the use of bispecific antibodies which bind to tumor antigen and a dendritic cell specific cell surface marker.
  • Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins which are expressed by the tumors and which are immunosuppressive. These include among others TGF- ⁇ (Kehrl etal. (1986) J. Exp. Med. 163: 1037-1050) , IL-10 (Howard &O'Garra (1992) Immunology Today 13: 198-200) , and Fas ligand (Hahne et al. (1996) Science 4: 1363-1365) . Antibodies to each of these entities can be used in combination with anti-LAG-3 to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
  • Anti-CD40 antibodies are able to substitute effectively for T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with LAG-3 antibodies (Ito et al. (2000) Immunobiology201 (5) 527-40) .
  • Activating antibodies toT cell costimulatory molecules such as CTLA-4 (e.g., US Patent No. 5,811,097) , OX-40 (Weinberg et al. (2000) Immunol. 164: 2160-2169) , 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997) , and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide for increased levels of T cell activation.
  • CTLA-4 e.g., US Patent No. 5,811,097
  • OX-40 Weinberg et al. (2000) Immunol. 164: 2160-2169
  • 4-1BB Melero
  • Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. While graft versus host disease is a consequence of this treatment, therapeutic benefit may be obtained from graft vs. tumor responses.
  • LAG-3 blockade can be used to increase the effectiveness of the donor engrafted tumor specific T cells.
  • another aspect of the invention provides a method of treating an infectious disease in a subject comprising administering to the subject an anti-LAG-3 antibody, or antigen-binding portion thereof, such that the subject is treated for the infectious disease.
  • the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) .
  • the antibody can be a chimeric or humanized antibody.
  • antibody mediated LAG-3 blockade can be used alone, or as an adjuvant, in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens.
  • pathogens for which this therapeutic approach can be particularly useful include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to HIV, Hepatitis (A, B, &C) , Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • LAG-3 blockade is particularly useful against established infections by agents such as HIV that present altered antigens over the course of the infections. These novel epitopes are recognized as foreign at the time of anti-human LAG-3 administration, thus provoking a strong T cell response that is not dampened by negative signals through LAG-3.
  • pathogenic viruses causing infections treatable by methods ofthe invention include HIV, hepatitis (A, B, or C) , herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-11, and CMV, Epstein Barr virus) , adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTL-V virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JCvirus and arboviral encephalitis virus.
  • herpes virus e.g., VZV, HSV-1, HAV-6, HSV-11, and CMV, Epstein Barr virus
  • adenovirus e.g., influenza virus, flaviviruses, echovirus, rhinovirus, coxsack
  • pathogenic bacteria causing infections treatable by methods ofthe invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
  • pathogenic fungi causing infections treatable by methods of the invention include Candida albicans, krusei, glabrata, tropicalis, etc. ) , Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc. ) , Genus Mucorales (mucor, absidia, rhizopus) , Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.
  • LAG-3 blockade can be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2) , or bispecific antibody therapy, which provides for enhanced presentation of tumor antigens (see, e.g., Bolliger (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123) .
  • cytokine treatment e.g., interferons, GM-CSF, G-CSF, IL-2
  • bispecific antibody therapy which provides for enhanced presentation of tumor antigens
  • Anti-LAG-3 antibodies may provoke and amplify autoimmune responses. Indeed, induction of anti-tumor responses using tumor cell and peptide vaccines revealsthat many anti-tumor responses involve anti-self reactivities (van Elsas et al. (2001) J. 112 Exp. Med. 194: 481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg &White (1996) J. Immunother Emphasis Tumor Immunol. 19 (1) : 81-4) .
  • anti-LAG-3 blockade in conjunction with various self-proteins in order to devise vaccination protocols to efficiently generate immune responses against these self-proteins for disease treatment.
  • Alzheimer's disease involves inappropriate accumulation of ⁇ peptide in amyloid deposits in the brain; antibody responses against amyloid are able to clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177) .
  • Analogous methods as described above for the use of anti-LAG-3 antibody can be used for induction of therapeutic autoimmune responses to treat patients having an inappropriate accumulation of other self-antigens, such as amyloid deposits, including ⁇ in Alzheimer's disease, cytokines such as TNF ⁇ , and IgE.
  • Anti-LAG-3 antibodies can be used to stimulate antigen-specific immune responses by co-administration of an anti-LAG-3 antibody with an antigen of interest (e.g., a vaccine) .
  • an antigen of interest e.g., a vaccine
  • the invention provides a method of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen; and (ii) an anti-LAG-3 antibody, or antigen-binding portion thereof, such that an immune response to the antigen in the subject is enhanced.
  • the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) .
  • the antibody can be a chimeric or humanized antibody.
  • the antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen.
  • antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria or other pathogens described above.
  • Suitable routes of administering the antibody compositions e.g., human monoclonal antibodies, multi-specific and bispecific molecules and immunoconjugates
  • the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous) .
  • Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
  • human anti-LAG-3 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent.
  • the antibody can be linked to the agent (as an immuno-complex) or can be administered separate from the agent. In the latter case (separate administration) , the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation.
  • Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin) , cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
  • anti-neoplastic agents such as doxorubicin (adriamycin) , cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
  • Cisplatin is intravenously administered as a 100 mg/mL dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/mL dose once every 21 days.
  • Co-administration of the human anti-LAG-3 antibodies, or antigen binding fragments thereof, of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.
  • kits comprising the antibody compositions of the invention (e.g., human antibodies, bispecific or multi-specific molecules, or immunoconjugates) and instructions for use.
  • the kit can further contain at least one additional reagent, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope inLAG-3 antigen distinct from the first human antibody) .
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • the progressive disorder of the nervous system that affects movement is Parkinson’s disease.
  • another aspect of the invention provides a method of treating Parkinson’s disease in a subject comprising administering to the subject an anti-LAG-3 antibody, or antigen-binding portion thereof, such that the subject is treated for Parkinson’s disease.
  • the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) .
  • the antibody can be a chimeric or humanized antibody.
  • LAG3 is enriched in the brain as well (C.J. Workman (2002) , Eur. J. Immunol. 32, 2255–2263) . Immunoblot analysis indicates that LAG3 is expressed predominantly in neurons. According to the Allen Brain Atlas, LAG3 is localized to neurons throughout the central nervous system (CNS) , including DA neurons. X. Mao et al., (Science 353, aah3374) reported that LAG3 preferentially binds ⁇ -synuclein ( ⁇ -syn) misfolded preformed fibrils (PFF) with high affinity mainly through its D1 domain (29-167AA) .
  • ⁇ -syn ⁇ -synuclein
  • PFF preformed fibrils
  • deletion of the D2 (168-252AA) , D3 (265-343AA) , or intracellular domain (ICD, 472-525AA) substantially weakens binding of LAG3 to ⁇ -syn PFF, X.
  • Mao et al have shown that ⁇ -syn PFF binding to LAG3 initiated a-syn PFF endocytosis, transmission, and toxicity.
  • Emerging evidence indicates that the pathogenesis of Parkinson’s disease (PD) may be due to cell-to-cell transmission of misfolded ⁇ -syn PFF.
  • Parkinson’s disease (PD) is the second most common neurodegenerative disorder and leads to slowness of movement, tremor, rigidity, and, in the later stages of PD, cognitive impairment.
  • PD is characterized by the accumulation of a-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system, with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD.
  • Anti-LAG3 antibody specifically bind to D1 or D2 domain can reduce a-syn PFF toxicity and cell-to-cell transmission, suggesting its potential for PD therapy. As shown in the Example 1, our antibody can specifically bind to D1 or D2 domain of LAG3 protein. Therefore, there antibody can be used for the PD therapy.
  • the invention provides methods of combination therapy in which an anti-LAG-3 antibody is co-administered with one or more additional antibodies that are effective in stimulating immune responses to thereby further enhance, stimulate or upregulate immune responses in a subject.
  • the invention provides a method for stimulating an immune response in a subject comprising administering to the subject an anti-LAG-3 antibody and one or more additional immunostimulatory antibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody, such that an immune response is stimulated in the subject, for example to inhibit tumor growth or to stimulate an anti-viral response.
  • the subject is administered an anti-LAG-3 antibody and an anti-PD-1 antibody.
  • the subject is administered an anti-LAG-3 antibody and an anti-PD-L1 antibody.
  • the subject is administered ananti-LAG-3 antibody and an anti-CTLA-4 antibody.
  • the anti-LAG-3 antibody is a human antibody, such as an antibody of the disclosure.
  • theanti-LAG-3 antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse anti-LAG-3 mAb) .
  • the at least oneadditional immunostimulatory antibody e.g., anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody
  • the at least one additional immunostimulatory antibody is a human antibody.
  • the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody) .
  • the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a CTLA-4 antibody to a subject.
  • a hyperproliferative disease e.g., cancer
  • the anti-LAG-3 antibody is administered at a subtherapeutic dose
  • the anti-CTLA-4 antibody is administered at a subtherapeutic dose
  • the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject.
  • the subject is human.
  • the anti-CTLA-4 antibody is human sequence monoclonal antibody 10D1 (described in PCT Publication WO 01114424) and the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein.
  • Other anti-CTLA-4 antibodies encompassed by the methods of the present invention include, for example, those disclosed in: WO98/42752; WO 00/37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95 (17) : 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22 (145) : Abstract No.
  • the anti-CTLA-4 antibody binds to human CTLA-4 with a K D of 5 x 10 -8 M or less, binds to human CTLA-4 with a K D of 1 x 10 -8 M or less, binds to human CTLA-4 with a K D of 5 x 10 -9 M or less, or binds to human CTLA-4 with a K D of between 1 x 10 -8 M and 1 x 10 -10 M or less.
  • the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a PD-1 antibody to a subject.
  • a hyperproliferative disease e.g., cancer
  • the anti-LAG-3 antibody is administered at a subtherapeutic dose
  • the anti-PD-1 antibody is administered at a subtherapeutic dose
  • the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-1 antibody to a subject.
  • the subject is human.
  • the anti-PD-1 antibody is a human sequence monoclonal antibody
  • the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein.
  • human sequence antiPD-1 antibodies include 17D8, 2D3, 4H1, 5C4 and 4A11, which are described in PCT Publication WO 061121168.
  • the anti-PD-1 antibody binds to human PD-1 with a K D of 5 x 10 -8 M or less, binds to human PD-1 with a K D of 1 x 10 -8 M or less, binds to human PD-1 with a K D of 5 x 10 -9 M or less, or binds to human PD-1with a K D of between 1 X 10 -8 M and 1 X 10 -10 M or less.
  • the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a PD-L1 antibody to a subject.
  • a hyperproliferative disease e.g., cancer
  • the anti-LAG-3 antibody is administered at a subtherapeutic dose
  • the anti-PD-L1 antibody is administered at a subtherapeutic dose
  • the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-L1 antibody to a subject.
  • the subject is human.
  • the anti-PD-L1 antibody is a human sequence monoclonal antibody and the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein.
  • human sequence anti-PD-L1 antibodies include 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7and 13G4, which are described in PCT Publication WO 07/005874.
  • the anti-PD-L1 antibody binds to human PD-L1 with a K D of 5 x 10 -8 M or less, binds to human PD-L1 with a K D of 1 x 10 -8 M or less, binds to human PD-L1with a K D of 5 x 10 -9 M or less, or binds to human PD-L1 with a K D of between 1 x 10 -8 M and 1 x 10 -10 M or less.
  • Blockade of LAG-3 and one or more second target antigens such as CTLA-4 and/or PD-1 and/or PD-L1 by antibodies can enhance the immune response to cancerous cells in the patient.
  • Cancers whose growth may be inhibited using the antibodies of the instant disclosure include cancers typically responsive to immunotherapy.
  • Representative examples of cancers for treatment with the combination therapy of the instant disclosure include those cancers specifically listed above in the discussion of monotherapy with anti-LAG-3 antibodies.
  • the combination of therapeutic antibodies discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each antibody in a pharmaceutically acceptable carrier.
  • the combination of therapeutic antibodies can be administered sequentially.
  • an anti-CTLA-4antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-CTLA-4 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-CTLA-4 antibody second.
  • an anti-PD-1 antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-PD-1 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-PD-1 antibody second.
  • an anti-PD-L1 antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-PD-L1 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-PD-Ll antibody second.
  • sequential administrations can be combined with concurrent administrations, or any combination thereof.
  • first administration of a combination anti-CTLA-4 antibody and anti-LAG-3antibody can be concurrent
  • second administration can be sequential with anti-CTLA-4 first and anti-LAG-3 second
  • third administration can be sequential with anti-LAG-3 first and anti-CTLA-4 second, etc.
  • the first administration of a combination anti-PD-1 antibody and anti-LAG-3 antibody can be concurrent, the second administration can be sequential with anti-PD-1 first and anti-LAG-3 second, and the third administration can be sequential with anti-LAG-3 first andanti-PD-1 second, etc.
  • the first administration of a combination anti-PD-Ll antibody and anti-LAG-3 antibody can be concurrent, the second administration can be sequential with anti-PD-Ll first and anti-LAG-3 second, and the third administration can be sequential with anti-LAG-3 first and anti-PD-Ll second, etc.
  • Another representative dosing scheme can involve a first administration that is sequential with anti-LAG-3 first and anti-CTLA-4 (and/or anti-PD-1 and/or anti-PD-Ll) second, and subsequent administrations may be concurrent.
  • the combination of anti-LAG-3 and one or more additional antibodies can be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , cells, and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173: 4919-28) .
  • an immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , cells, and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173: 4919-28) .
  • Non-limiting examples of tumor vaccines that can beused include peptides of melanoma antigens, such as peptides of gpl00, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.
  • a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-Ll blockade can be further combined with a vaccination protocol, such as any of the vaccination protocols discussed in detail above with respect to monotherapy with anti-LAG-3 antibodies.
  • a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can also be further combined with standard cancer treatments.
  • a combined LAG-3and CTLA-4 and/or PD-1 and/or PD-L1 blockade can be effectively combined with chemotherapeutic regimes.
  • it is possible to reduce the dose of other chemotherapeutic reagent administered with the combination of the instant disclosure Mokyr et al. (1998) Cancer Research 58: 5301-5304) .
  • a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combination with decarbazine for the treatment of melanoma.
  • Another example is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combination with interleukin-2 (IL-2) for the treatment of melanoma.
  • IL-2 interleukin-2
  • LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade with chemotherapy are a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
  • Other combination therapies that may result in synergy with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade through cell death include radiation, surgery, or hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.
  • Angiogenesis inhibitors can also be combined with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade. Inhibition of angiogenesis leads to tumor cell death, which can be a source of tumor antigen fed into host antigen presentation pathways.
  • a combination of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blocking antibodies can also be used in combination with bispecific antibodies that target Fc ⁇ or Fc ⁇ receptor- expressing effector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5, 922, 845 and 5, 837, 243) .
  • Bispecific antibodies can be used to target two separate antigens. The T cell arm of these responses would be augmented by the use of a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade.
  • a combination of anti-LAG-3 and anti-CTLA-4 and/or antiPD-1 antibodies and/or anti-PD-L1 antibodies can be used in conjunction with anti-neoplastic antibodies, such as (rituximab) , (trastuzumab) , (tositumomab) , (ibritumomab) , (alemtuzumab) , (eprtuzumab) , (bevacizumab) , and (erlotinib) , and the like.
  • anti-neoplastic antibodies such as (rituximab) , (trastuzumab) , (tositumomab) , (ibritumomab) , (alemtuzumab) , (eprtuzumab) , (bevacizumab) , and (erlotinib) , and the like.
  • a treatment of a hyperproliferative disease can include an anti-cancer antibody in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies, concurrently or sequentially or any combination thereof, which can potentiate an anti-tumor immune responses by the host.
  • Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins, which are expressed by the tumors and which are immunosuppressive. These include, among others, TGF- ⁇ (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050) , IL-10 (Howard &O'Garra (1992) Immunology Today 13: 198-200) , and Fas ligand (Hahne et al. (1996) Science 27 4: 1363-1365) .
  • antibodies to each of these entities can be further combined with an anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibody combination to counteract the effects of immunosuppressive agents and favor anti-tumor immune responses by the host.
  • Anti-CD40 antibodies can be used in conjunction with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 combination (Ito et al., supra) .
  • Other activating antibodies to T cell co stimulatory molecules Weinberg et al., supra, Melero et al. supra, Hutloff et al., supra) may also provide for increased levels of T cell activation.
  • a combined LAG-3 and CTLA-4 and/orPD-1 and/or PD-L1 blockade can be used to increase the effectiveness of the donor engrafted tumor specific T cells.
  • the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of anti-CTLA-4 and/or anti-PD-1and/or anti-PD-L1 antibody to a subject.
  • a hyperproliferative disease e.g., cancer
  • the methods of the present invention provide for a method of reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to the patient. Because any patient who will receive an immunostimulatory therapeutic antibody is at risk for developing colitis or diarrhea induced by such an antibody, this entire patient population is suitable for therapy according to the methods of the present invention.
  • steroids have been administered to treat inflammatory bowel disease (IBD) and prevent exacerbations of IBD, they have not been used to prevent (decrease the incidence of) IBD in patients who have not been diagnosed with IBD.
  • IBD inflammatory bowel disease
  • a combination LAG-3 and CTLA-4 and/or PD-1 and/orPD-L1 blockade i.e., immunostimulatory therapeutic antibodies anti-LAG-3 and antiCTLA-4 and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies
  • a "nonabsorbable steroid" is a glucocorticoid that exhibits extensive first pass metabolism such that, following metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20%.
  • the non-absorbable steroid isbudesonide.
  • Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration.
  • ENTOCORT (Astra-Zeneca) is a pH-and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and throughout the colon.
  • ENTOCORT is approved in the U.S. for the treatment of mild to moderate Crohn's disease involving theileum and/or ascending colon.
  • the usual oral dosage of ENTOCORT for thetreatment of Crohn's disease is 6 to 9 mg/day.
  • ENTOCORT is released in the intestines before being absorbed and retained in the gut mucosa.
  • ENTOCORT is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (about 10%) .
  • the low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less extensive first-pass metabolism.
  • Budesonide results in fewer adverse effects, including less hypothalamic-pituitary suppression, than systemically-acting corticosteroids.
  • chronic administration of ENTOCORT can result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR 58th ed.2004; 608-610.
  • a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade i.e., immunostimulatory therapeutic antibodies anti-LAG-3 andanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies
  • a nonabsorbable steroid in conjunction with a nonabsorbable steroid can be further combined with a salicylate.
  • Salicylates include 5-ASAagents such as, for example: sulfasalazine ( Pharmacia &UpJohn) ; olsalazine ( Pharmacia &UpJohn) ; balsalazide ( Salix Pharmaceuticals, Inc. ) ; and mesalamine ( Procter &Gamble Pharmaceuticals; Shire US; Axcan Scandipharm, Inc.; ROW Solvay) .
  • a salicylate administered in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 antibodies and a non-absorbable steroid can include any overlapping or sequential administration of the salicylate and the non-absorbable steroid for the purpose of decreasing the incidence of colitis induced by the immunostimulatory antibodies.
  • methods for reducing the incidence of colitis induced by the immunostimulatory antibodies according to the present invention encompass administering a salicylate and a non-absorbable concurrently or sequentially (e.g., a salicylate is administered 6 hours after a non-absorbable steroid) , or any combination thereof.
  • a salicylate and a non-absorbable steroid can be administered by the same route (e.g., both are administered orally) or by different routes (e.g., a salicylate is administered orally and a non-absorbable steroid is administered rectally) , which may differ from the route (s) used to administer the anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies.
  • Anti-LAG3 human monoclonal antibodies were generated by screening full human Fab phage-display libraries. Wildtype LAG-3-ECD-huFc fragments can bind to Daudi cells while D1-D2 truncated LAG-3-ECD-huFc fragments fail to bind Daudi cells. Consequently, the D1-D2 domains are critical for LAG-3 function.
  • LAG-3 is a single-pass type I membrane protein which belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains (ECD) : domain (D) 1, D2, D3 and D4.
  • ECD extracellular Ig-like domains
  • a recombinant human LAG-3-ECD-human IgG1 (LAG-3-huFc) fusion protein or a human D1-D2 truncated LAG-3-ECD-human IgG1 ( ⁇ D1D2-LAG-3-huFc) fusion protein were expressed in a 293T cell system.
  • Phage library Ig gene segments in mammals are arranged in groups of variable (V) , diversity (D) , joining (J) , and constant (C) exons.
  • the human Fab phage library were construed using the phage vectors, which consists of: 1) all human variable kappa (VK) repertoires; and 2) the VH of VH3-23 and VH1-69 germline genes, respectively, with genetically randomized CDR3 regions from healthy human subjects.
  • 293F cells were transfected with a plasmid containing a D1-D2 deleted LAG-3 ( ⁇ D1D2-LAG-3) sequence with a FLAG-tag at the N-terminus.
  • ⁇ D1D2-LAG-3 293F cells were used for phage library screening.
  • the phage libraries were performed the sequential negative screenings: streptavidin beads, ⁇ D1D2-LAG-3 transfected 293F cells and biotin-labeled-human IgG1Fc protein.
  • the phage libraries were performed the sequential negative screenings: casein-coated immunotubes, ⁇ D1D2-LAG-3 transfected 293F cells and human IgG1Fc protein.
  • the resulting library was then incubated in LAG3-huFc-coated immunotubes for 2 hrs under motion. Unbound phages were removed by washing with PBST 5-20 times. Similar with cell based panning, three rounds of phage library screening were performed in total.
  • Output-3 phage libraries were diluted and plated to grow at 37°C for 8 hrs and captured by anti-kappa antibody-coated filters overnight at 22°C.
  • Biotinylated LAG-3-huFc (50nM) and NeutrAvidin-AP conjugate were applied to the filter to detect antigen binding anti-LAG3 phages. Positive phage plaques were picked and eluted into 100 ⁇ L of phage elution buffer.
  • eluted phages were then used to infect 1 mL of XL1-Blue competent cells to make a high-titer (HT) phage for phage single point ELISA (SPE) (ELISA immobilized substrate coated with 50 nM of each protein tested) . 1x10 10 plaque forming units (pfus) of each phage hit was used for SPE confirmation.
  • the positive clones picked from the filter lift were then tested for LAG-3 antigen binding with LAG-3-huFc and ⁇ D1D2-LAG-3-huFc.
  • the D1-D2 specific binders were amplified from antigen positive phages by PCR and sequenced. Ig light chain V genes (VL) and VH sequences were analyzed to identify unique sequences and determine sequence diversity.
  • VL and VH gene sequences of all hits were cloned into expression vectors pFUSE2ss-CLIg-hk (light chain, InvivoGen Cat No. pfuse2ss-hclk) and pFUSEss-CHIg-hG1 (heavy chain, InvivoGen Cat No. pfusess-hchg1) .
  • the antibodies were expressed in HEK293 cells and purified using Protein A PLUS-Agarose.
  • Example 1 To evaluate the capability of the anti-LAG-3 antibodies to bind to human, rat, and mouse LAG3 the antibodies identified in Example 1 were evaluated for their binding property through ELISA.
  • the human, rat and mouse LAG3ECD-Fc protein were coated to ELISA plate at 1 ⁇ g/ml with 100 ⁇ l/well.
  • Antibodies from Example 1 were serially diluted with ELISA diluent buffer.
  • LAG-3 antibodies at various concentrations 10 ⁇ g/ml, 3.333 ⁇ g/ml, 1.111 ⁇ g/ml, 0.370 ⁇ g/ml, 0.123 ⁇ g/ml, 0.041 ⁇ g/ml, 0.014 ⁇ g/ml, 0.005 ⁇ g/ml, 0.0015 ⁇ g/ml and 0.0005 ⁇ g/ml) were then added to LAG3 antigen coated plate for 1.5hr RT. The resulting plates were washed and then labeled with anti-human IgG (Fab) -HRP antibody.
  • the S31 can only bind to human LAG3.
  • the S27 and T99 can bind to human LAG3 and rat/mouse LAG3 with lower potency.
  • the S119 antibody can bind to human, rat and mouse LAG3 at high potency (FIG. 2) .
  • LAG-3 is expressed on activated or exhausted T cells.
  • CD4+ T cells were isolated using CD4 magnetic beads. The purified human CD4+ T cells were stimulated with Human T-Activator CD3/CD28 for 72 hrs. Antibodies from Example 1 were serially diluted with FACS buffer.
  • LAG-3 antibodies at various concentrations (10 ⁇ g/ml, 3.333 ⁇ g/ml, 1.111 ⁇ g/ml, 0.370 ⁇ g/ml, 0.123 ⁇ g/ml, 0.041 ⁇ g/ml, 0.014 ⁇ g/ml and 0.005 ⁇ g/ml) were then added to the activated human CD4 T cells in the presence of mouse anti-human LAG3 PE antibody (eBioscience, clone: 3DS223H) for 30 min on ice. The labeled cells were washed with FACS buffer and subsequently labeled with APC-conjugated anti-human IgG antibodies for 30 min on ice. The resulting cells were washed once with FACS buffer.
  • mouse anti-human LAG3 PE antibody eBioscience, clone: 3DS223H
  • Labeled cells were evaluated for fluorescence intensity by flow cytometry in a BD FACSCalibur TM . As shown in FIG. 3, the S27, S31, T99 and S119 can dose-dependent binding to LAG3 expressed on the activated human CD4+ T cells.
  • Anti-LAG-3 antibody inhibition of soluble LAG-3 (sLAG) binding to MHC class II receptor Anti-LAG-3 antibody inhibition of soluble LAG-3 (sLAG) binding to MHC class II receptor.
  • an in vitro binding assay was designed using biotin-labeled LAG-3-ECD-huFcfusion proteins and Raji cells expressing MHC class II receptor.
  • Antibodies from Example 1 were serially diluted from 20 ⁇ g/mL with FACS buffer and pre-incubated with 6 ⁇ g/mL of biotin-LAG-3-ECD-huFcc for 30 min at room temperature. The antibody mixture was then added to FcR blocked Raji cells and incubated for 30 min on ice.
  • PBMCs peripheral blood mononuclear cells
  • Staphylococcal enterotoxin B is a superantigen that simultaneously bindings to MHC class II antigens and T cell receptors (TCRs) , bringing them together in such a way as to induce T cell proliferation and cytokine production.
  • 2 ⁇ 10 5 PBMCs were stimulated with SEB in the presence of the antibodies from Example 1 at various concentrations starting from 20 ⁇ g/ml at 1: 3 serious dilution for 6 doses.
  • IL-2 concentration in the culture supernatant was evaluated by ELISA.
  • the anti-LAG3 antibodies S24, S27, S31, S87, S119, T99 and S20 can dose dependently enhance the IL-2 production as compared with SEB stimulation only.
  • T regs regulatory T cells
  • T effs effector T cells
  • T regs CD4+CD25hi
  • mediates their suppressive function Journal of Immunology 184: 6545-51, 2010
  • T regs CD4 + CD25 - CD127 hi
  • T effs CD4 + CD25 - CD127 hi
  • T effs were then labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with T regs at a 1: 1 ratio in the presence of plate bound anti-CD3 antibodies and mitomycin C-treated antigen presenting cells.
  • Anti-LAG-3 antibodies were next added to the cell culture and T effs cell proliferation were tested 5 days later.
  • the results in FIG. 6, indicate that when Tregs were co-cultured with effector T cells, effector T cell proliferation and cytokine production was inhibited. S119 and T99 can reverse the inhibition of Teffs by Tregs.
  • Staphylococcal enterotoxin B is a superantigen that stimulate the human immune response.
  • PD-1 blocking antibody can enhance the SEB stimulated IL-2 production.
  • anti-LAG3 antibodies can also highlight the SEB mediated IL-2 production.
  • To explore the effect of an anti-LAG3 antibody in combination with PD-1 antibody we investigated the effect of anti-LAG3 antibody on SEB stimulation in the presence of suboptimal PD-1 stimulation. In the presence of 0.1 ⁇ g/ml PD-1 antibody, the serious diluted anti-LAG3 antibodies were added to the SEB culture. IL-2 production were evaluated 72hr later. The results in FIG. 7 indicate that anti-LAG-3 antibodies can enhance the SEB stimulated T cell response in a dose-dependent manner in the presence of suboptimal PD-1 treatment, suggesting that anti-LAG3 and anti-PD-1 combo treatment have synergistic effect.
  • Anti-LAG-3 antibodies enhance human T cell response in the presence of PD-L1 antibody.
  • LAG-3 antibody synergy with PD-1/PD-L1 blockade
  • NSG mice NOD scid gamma deficient and the most immunodeficient mice making them ideal recipients for human tumor cell and PBMC grafting.
  • 10 days post-graft human PBMCs will be transplanted into the tumor-bearing mice.
  • PD-1/PD-L1 antibodies (2mg/kg) alone, or PD-L1 and LAG-3 (10mg/kg for each LAG3 antibody) antibodies together, will be administered to the mice every other day. Tumor volume will be monitored every other day in conjunction with antibody administration.
  • the 2mg/kg PD-L1/PD-1 antibody will show minimal effect on tumor volume.
  • the LAG3 combo will significantly shrink the tumor volume.
  • the binding of the S20, S24, S27, S31, S87, S119, S120, S128, S136, S161 and T99 antibodies to recombinant his-taq human LAG3-ECD protein was examined by BIAcore T200 using a capture method.
  • the anti-LAG3 antibodies were captured using anti-human Fc antibody.
  • the anti-human Fc antibody was coated on chip.
  • the serious concentration of his-taq human LAG3-ECD protein (0-4nM) were injected over capture antibodies at the flow rate of 30 ⁇ l/min.
  • the dissociation phase were 900s or 550s.
  • Table 1 The results are shown in Table 1 below.
  • the Biacore results for the anti-LAG3 antibodies have shown that these anti-LAG3 antibodies are high affinity binder to human LAG3.

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Abstract

The present invention provides antibodies that bind Lymphocyte Activation Gene-3 (LAG-3). Also provided are methods of stimulating an immune response, inhibiting growth of tumor cells, and treating an autoimmune, inflammatory, or viral disease.

Description

ANTI-LYMPHOCYTE ACTIVATION GENE-3 (LAG-3) ANTIBODIES AND USES THEREOF FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and protein biochemistry. More specifically, the invention relates to antibodies that bind to Lymphocyte Activation Gene-3 (LAG-3) and methods of use thereof.
BACKGROUND
Lymphocyte Activation Gene-3 (LAG-3) (also known as CD223) is a member of the immunoglobulin (Ig) superfamily, is closely related to CD4, and variously impacts T cell function. LAG-3 is expressed on activated T cells, exhausted T cells, tumor infiltrating T cells, and regulatory T cells (Tregs) . Upon binding with major histocompatibility complex 2 (MHC class II) , the LAG-3/MHC class II interaction results in the negative regulation of T cell proliferation, activation, and homeostasis.
LAG-3 represents an important immune checkpoint in cancer, similarly to cytotoxic T lymphocyte antigen-4 (CTLA-4) , programmed cell death ligand-1 (PD-L1) , and programmed cell death-1 (PD-1) . LAG-3 not only expresses on the activated/exhausted effector T cells but also on regulatory T cells. LAG3 antagonism can not only promote the activation of effector T cells, but also block the suppressive function of regulatory T cells. Therefore, LAG-3 represents a promising target for cancer immunotherapy and preclinical evidence suggests that an anti-LAG-3 antibody can promote an anti-tumor response.
In view of the above, a need exists for developing novel agents that modulate the activity of LAG-3 in a manner that stimulates an immune response that inhibits the growth of various cancers and tumor cells, as well as being useful in the treatment of autoimmune, inflammatory, or viral diseases.
SUMMARY OF THE INVENTION
The present disclosure describes anti-LAG-3 mAbs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following:
i. binds human LAG-3；
ii. blocks LAG-3 binding to major histocompatibility complex (MHC) class II molecules；
iii. stimulates an immune response； and；
iv. reverses the inhibitory effect of regulatory T cells on effector cells.
The antibodies of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with LAG-3 in humans and animals, including the prevention and treatment of solid and hematological cancers. The antibodies of the disclosure are also useful as diagnostics to determine the level of LAG-3 expression in tissue samples. Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof； pharmaceutical compositions comprising one or more of the anti-LAG-3 monoclonal antibodies, preferably chimeric or humanized forms of said antibodies； methods of therapeutic use of such anti-LAG-3 monoclonal antibodies； and cell lines that produce these anti-LAG-3 monoclonal antibodies.
The embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined by reference to specific structural characteristics i.e. specified amino acid sequences of either the CDRs or entire heavy chain or light chain variable domains. All of these antibodies bind to LAG-3.
The monoclonal antibodies, or antigen binding fragments thereof may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide. In some embodiments, an antibody comprises at least one light chain comprising the three light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy chain comprising the three heavy chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.
Preferred embodiments are anti-LAG-3 mAbs, or antigen binding fragments thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2； said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 4； and said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID  NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45. The heavy chain variable domain may comprise any one of the listed variable heavy chain CDR1 sequences (HCDR1) in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR3 sequences (HCDR3) . However, certain embodiments of HCDR1 and HCDR2 and HCDR3 are particularly preferred, which derive from a single common VH domain, examples of which are described herein.
The antibody or antigen binding fragment thereof may additionally comprise a light chain variable domain (VL) , which is paired with the VH domain to form an antigen binding domain. Preferred light chain variable domains are those comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 115； said variable light chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 SEQ ID NO: 102, and SEQ ID NO: 103； and said variable light chain CDR3 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID  NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139.
The light chain variable domain may comprise any one of the listed variable light chain CDR1 sequences (LCDR1) in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR3 sequences (LCDR3) . However, certain embodiments of LCDR1 and LCDR2 and LCDR3 are particularly preferred, which derive from a single common VL domain, examples of which are described herein.
Any given LAG-3 antibody or antigen binding fragment thereof comprising a VH domain paired with a VL domain will comprise a combination of 6 CDRs: variable heavy chain CDR1 (HCDR1) , variable heavy chain CDR2 (HCDR2) , variable heavy chain CDR3 (HCDR3) , variable light chain CDR1 (LCDR1) , variable light chain CDR2 (LCDR2) , and variable light chain CDR1 (LCDR1) . Although all combinations of 6 CDRs selected from the CDR sequence groups listed above are permissible, and within the scope of the disclosure, certain combinations of 6 CDRs are particularly preferred.
Preferred combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDR1 (HCDR1) , variable heavy chai n CDR2 (HCDR2) , variable heavy chain CDR3 (HCDR3) , variable light chain CDR1 (LCDR1) , variable light chain CDR2 (LCDR2) , and variable light chain CDR3 (LCDR3) selected from the group consisting of:
i. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 5, LCDR1 comprising SEQ ID NO: 46, LCDR2 comprising SEQ ID NO: 81, LCDR3 comprising SEQ ID NO: 104；
ii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 6, LCDR1 comprising SEQ ID NO: 47, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105；
iii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 7, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106；
iv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 8, LCDR1 comprising SEQ ID NO: 49, LCDR2 comprising SEQ ID NO: 83, LCDR3 comprising SEQ ID NO: 105；
v. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 9, LCDR1 comprising SEQ ID NO: 50, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106；
vi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 107；
vii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 11, LCDR1 comprising SEQ ID NO: 51, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 108；
viii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 109；
ix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 13, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 104；
x. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 14, LCDR1 comprising SEQ ID NO: 53, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 110；
xi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
xii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 16, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105；
xiii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 17, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112；
xiv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 18, LCDR1 comprising SEQ ID NO: 56, LCDR2 comprising SEQ ID NO: 87, LCDR3 comprising SEQ ID NO: 113；
xv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
xvi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 19, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112；
xvii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 20, LCDR1 comprising SEQ ID NO: 57, LCDR2 comprising SEQ ID NO: 98, LCDR3 comprising SEQ ID NO: 114；
xviii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 115；
xix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 22, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 116；
xx. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 23, LCDR1 comprising SEQ ID NO: 59, LCDR2 comprising SEQ ID NO: 88, LCDR3 comprising SEQ ID NO: 117；
xxi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 24, LCDR1 comprising SEQ ID NO: 60, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
xxii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 25, LCDR1 comprising SEQ ID NO: 61, LCDR2 comprising SEQ ID NO: 89, LCDR3 comprising SEQ ID NO: 118；
xxiii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119；
xxiv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 27, LCDR1 comprising SEQ ID NO: 63, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 111；
xxv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119；
xxvi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
xxvii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 28, LCDR1 comprising SEQ ID NO: 65, LCDR2 comprising SEQ ID NO: 91, LCDR3 comprising SEQ ID NO: 120；
xxviii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 29, LCDR1 comprising SEQ ID NO: 66, LCDR2 comprising SEQ ID NO: 92, LCDR3 comprising SEQ ID NO: 121；
xxix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 30, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 93, LCDR3 comprising SEQ ID NO: 122；
xxx. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 67, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 123；
xxxi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 32, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 124；
xxxii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 33, LCDR1 comprising SEQ ID NO: 68, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 125；
xxxiii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 34, LCDR1 comprising SEQ ID NO: 69, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 126；
xxxiv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 35, LCDR1 comprising SEQ ID NO: 70, LCDR2 comprising SEQ ID NO: 94, LCDR3 comprising SEQ ID NO: 127；
xxxv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 128；
xxxvi. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 36, LCDR1 comprising SEQ ID NO: 71, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 129；
xxxvii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 115, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 130；
xxxviii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 34, LCDR1 comprising SEQ ID NO: 70, LCDR2 comprising SEQ ID NO: 96, LCDR3 comprising SEQ ID NO: 131；
xxxix. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 37, LCDR1 comprising SEQ ID NO: 72, LCDR2 comprising SEQ ID NO: 97, LCDR3 comprising SEQ ID NO: 132；
xl. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 38, LCDR1 comprising SEQ ID NO: 72, LCDR2 comprising SEQ ID NO: 97, LCDR3 comprising SEQ ID NO: 132；
xli. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 39, LCDR1 comprising SEQ ID NO: 73, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 133；
xlii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 40, LCDR1 comprising SEQ ID NO: 74, LCDR2 comprising SEQ ID NO: 99, LCDR3 comprising SEQ ID NO: 134；
xliii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 41, LCDR1 comprising SEQ ID NO: 75, LCDR2 comprising SEQ ID NO: 93, LCDR3 comprising SEQ ID NO: 135；
xliv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 76, LCDR2 comprising SEQ ID NO: 100, LCDR3 comprising SEQ ID NO: 130；
xlv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 42, LCDR1 comprising SEQ ID NO: 76, LCDR2 comprising SEQ ID NO: 100, LCDR3 comprising SEQ ID NO: 136；
xlvi. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 43, LCDR1 comprising SEQ ID NO: 77, LCDR2 comprising SEQ ID NO: 101, LCDR3 comprising SEQ ID NO: 137；
xlvii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 35, LCDR1 comprising SEQ ID NO: 78, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 138；
xlviii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 44, LCDR1 comprising SEQ ID NO: 79, LCDR2 comprising SEQ ID NO: 102, LCDR3 comprising SEQ ID NO: 122； and
xlix. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 45, LCDR1 comprising SEQ ID NO: 80, LCDR2 comprising SEQ ID NO: 103, LCDR3 comprising SEQ ID NO: 139.
Further preferred anti-LAG-3 antibodies include antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, and SEQ ID NO: 188, and amino acid sequences exhibiting at least 90％, 95％, 97％,  98％, or 99％sequence identity to one of the recited sequences. Alternatively or in addition, preferred anti-LAG-3 antibodies including antibodies or antigen binding fragments thereof may comprise a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, and SEQ ID NO: 237, and amino acid sequences exhibiting at least 90％, 95％, 97％, 98％, or 99％sequence identity to one of the recited sequences.
Although all possible pairing of VH domains and VL domains selected from the VH and VL domain sequence groups listed above are permissible, and within the scope of the disclosure, certain combinations of VH and VL domains are particularly preferred. Accordingly, preferred LAG-3 antibodies, or antigen binding fragments thereof, are those comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL) , wherein the combination is selected from the group consisting of:
i. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 140 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 189；
ii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 141 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 190；
iii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 142 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 191；
iv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 143 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 192；
v. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 144 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 193； 
vi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 194；
vii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 195；
viii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 147 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 196；
ix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 148 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 197；
x. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 149 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 198；
xi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 199；
xii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 151 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 200；
xiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 201；
xiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 153 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 202；
xv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 203；
xvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 204；
xvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 205；
xviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 206；
xix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 207；
xx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 208；
xxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 209；
xxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 210；
xxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 211；
xxiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 212；
xxv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 213；
xxvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 165 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 214；
xxvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 215；
xxviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 167 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 216；
xxix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 217；
xxx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 218；
xxxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 219；
xxxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 220；
xxxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 221；
xxxiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 173 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 222；
xxxv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 174 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 223；
xxxvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 175 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 224；
xxxvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 176 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 225；
xxxviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 226；
xxxix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 227；
xl. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 179 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 228；
xli. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 180 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 229；
xlii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 181 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 230；
xliii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 182 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 231；
xliv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 183 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 232；
xlv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 184 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 233；
xlvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 185 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 234；
xlvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 186 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 235；
xlviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 236； and
xlix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 237.
Preferred anti-LAG-3 antibodies or antigen binding fragments thereof may also comprise a combination of a heavy chain variable domain and a light chain variable domain wherein the  heavy chain variable domain comprises a VH sequence with at least 85％sequence identity, or at least 90％sequence identity, or at least 95％sequence identity, or at least 97％, 98％or 99％sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xlix) and/or the light chain variable domain comprises a VL sequence with at least 85％sequence identity, or at least 90％sequence identity, or at least 95％sequence identity, or at least 97％, 98％or 99％sequence identity, to the light chain amino acid sequences shown above in (i) to (xlix) . The specific VH and VL pairings or combinations in parts (i) through (xlix) may be preserved for anti-LAG-3 antibodies having VH and VL domain sequences with a particular percentage sequence identity to these reference sequences.
For all embodiments wherein the heavy chain and/or light chain variable domains of the antibodies or antigen binding fragments are defined by a particular percentage sequence identity to a reference sequence, the VH and/or VL domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
Preferred embodiments of the anti-LAG-3 antibodies described herein, are also characterized by combinations of properties which are not exhibited by prior art anti-LAG-3 antibodies proposed for human therapeutic use. Accordingly, the preferred anti-LAG-3 antibodies described herein are characterized by:
i. binds human LAG-3；
ii. blocks LAG-3 binding to major histocompatibility complex (MHC) class II molecules；
iii. stimulates an immune response； and
iv. reverses the inhibitory effect of regulatory T cells on effector cells.
In yet another preferred embodiment described herein, the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat LAG-3.
Various forms of the anti-LAG-3 mAbs disclosed are contemplated herein. For example, the anti-LAG-3 mAbs can be full length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgG1, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchangeor an antibody fragment, e.g., a F (ab') 2 fragment, a F (ab) fragment, a single chain Fv fragment (scFv) , etc., as disclosed herein.
The preferred embodiments of the disclosure provide pharmaceutical or veterinary compositions comprising one or more of the anti-LAG-3 mAbs or fragments disclosed herein, optionally chimeric or humanized forms, and a pharmaceutically acceptable carrier, diluent, or excipient.
Prior to the present disclosure, there was a need to identify anti-LAG-3 mAbs that possess the functional profiles as described herein. The anti-LAG-3 mAbs of the present disclosure exhibit distinct combinations of properties, particularly combinations of properties that render the mAbs particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers, ischemia-reperfusion injury, autoimmune and/or inflammatory diseases.
Further scope of the applicability of the present disclosure will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. The D1-D2 domains are critical for LAG-3 function. Wildtype (WT) LAG3 extracellular domain (ECD) fusion protein (LAG-3-ECD-huFc) fragments can bind to Daudi cells while D1-D2 truncated LAG-3-ECD-huFc fragments fail to bind Daudi cells.
FIG. 2. The binding of human anti-LAG3 antibodies to LAG3 protein derived from various species. Anti-LAG-3 antibodies were evaluated for their binding properties to human, rat, and mouse LAG3 through enzyme-linked immunosorbent assay (ELISA) .
FIG. 3. The binding of human anti-LAG3 antibodies to cell surface LAG-3 antigen on activated human primary CD4+ T cells. Anti-LAG-3 antibodies were assessed for binding to cell surface LAG-3 antigen on activated human primary CD4+ T cells at various concentrations (10 μg/ml, 3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123 μg/ml, 0.041 μg/ml, 0.014 μg/ml and 0.005 μg/ml) .
FIG. 4. Inhibition of soluble LAG-3 (sLAG) binding to MHC class II receptor by anti-LAG-3 antibody. Anti-LAG-3 antibodies were evaluated for their ability to block the binding of  sLAG-3 to MHC class II receptor in an in vitro binding assay using biotin-labeled LAG-3-ECD-huFcLAG-3-Fc fusion proteins and Raji cells expressing MHC class II receptor.
FIG. 5. Stimulation of IL-2 production in peripheral blood mononuclear cells (PBMCs) by anti-LAG-3 antibodies. Anti-LAG-3 antibodies were administrated into Staphylococcal Enterotoxin B (SEB) stimulated PBMC at various concentrations starting from 20μg/ml at 1: 3 serious dilution for 6 doses. Three days later, IL-2 concentration in the culture supernatant was evaluated by enzyme-linked immunosorbent assay (ELISA) .
FIG. 6. Reversing the suppressive function of regulatory T cells (Tregs) on effector T cells (Teffs) using anti-LAG-3 antibodies. To evaluate the ability of anti-LAG-3 antibodies to reverse the suppressive effect of Tregs on Teffs, the antibodies of Example 1 were used in an in vitro Tregs suppression assay.
FIG. 7. Synergistic effect of anti-LAG3 and PD-1 antibody combo treatment. The anti-LAG3 antibodies were tested in combination with PD-1 antibody on SEB-stimulated PBMCs assay.
FIG. 8. Anti-LAG-3 antibodies enhance human T cell response in the presence of PD-L1 antibody. The anti-. antibodies were evaluated in combination with PD-L1 antibody on human mixed lymphocyte reaction (MLR) assay.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, which bind to human LAG-3 and that have desirable functional properties. In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain germline sequences and/or comprise particular structural features such as CDR regions comprising particular amino acid sequences. This disclosure provides isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies and pharmaceutical compositions containing the antibodies, immunoconjugates or bispecific molecules of the invention. This disclosure also relates to methods of using the antibodies, such as to detect LAG-3 protein, as well as to methods of using the anti-LAG-3 antibodies of the invention to stimulate immune responses, alone or in combination with other immunostimulatory antibodies. Accordingly, this disclosure also provides methods of using the anti-LAG-3 antibodies of the invention to, for example, inhibit tumor growth or treat viral infection.
In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term "LAG-3" refers to Lymphocyte Activation Gene-3. The LAG3 protein, which belongs to immunoglobulin (Ig) superfamily, comprises a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains, designated D1 to D4. As described herein, the term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs. For example, antibodies specific for a human LAG-3 protein may, in certain cases, cross-react with a LAG-3 protein from a species other than human. In other embodiments, the antibodies specific for a human LAG-3 protein may be completely specific for the human LAG-3 protein and may not exhibit species or other types of cross-reactivity, or may cross-react with LAG-3 from certain other species but not all other species (e.g., cross-react with monkey LAG-3, but not mouse LAG-3) . The term "human LAG-3" refers to human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 having GenBank Accession No. NP 002277. The term "mouse LAG-3" refers to mouse sequence LAG-3, such as the complete amino acid sequence of mouse LAG-3 having GenBank Accession No. NP 032505. LAG-3 is also known in the art as, for example, CD223. The human LAG-3 sequence may differ from human LAG-3 of GenBank Accession No. NP 002277 by having, e.g., conserved mutations or mutations in non-conserved regions and the LAG-3 has substantially the same biological function as the human LAG-3 of GenBank Accession No. NP 002277. For example, a biological function of human LAG-3 is having an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody of the instant disclosure or a biological function of human LAG-3 is binding to MHC Class II molecules.
A particular human LAG-3 sequence will generally be at least 90％identical in amino acids sequence to human LAG-3 of GenBank Accession No. NP 002277 and contains amino acid residues that identify the amino acid sequence as being human when compared to LAG-3 amino acid sequences of other species (e.g., murine) . In certain cases, a human LAG-3 can be at least 95％, or even at least 96％, 97％, 98％, or 99％identical in amino acid sequence to LAG-3 of GenBank Accession No. NP 002277. In certain embodiments, a human LAG-3 sequence will display no more than 10 amino acid differences from the LAG-3 sequence of GenBank Accession No. NP 002277. In certain embodiments, the human LAG-3 can display no more than  5, or even no more than 4, 3, 2, or 1 amino acid difference from the LAG-3 sequence of GenBank Accession No. NP 002277. Percent identity can be determined as described herein.
The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
An "antigen-specific T cell response" refers to responses by a T cell that result from stimulation of the T cell with the antigen for which the T cell is specific. Non-limiting examples of responses by a T cell upon antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production) .
The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion" ) or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C H1, CH2, and C H3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FR) . Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
The term "antigen-binding portion" of an antibody (or simply "antibody portion" ) , as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a LAG-3 protein) . It has been shown that the antigen-binding function  of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and C H1 domains； (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region； (iii) a Fab'fragment, which is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3. sup. rd ed. I993) ； (iv) a Fd fragment consisting of the VH and C H1 domains； (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a VH domain； (vii) an isolated complementarity determining region (CDR) ； and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ； see e.g., Bird et al. (1988) Science 242: 423-426； and Huston et al. (I988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) . Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
An "isolated antibody" , as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a LAG-3 protein is substantially free of antibodies that specifically bind antigens other than LAG-3 proteins) . An isolated antibody that specifically binds a human LAG-3 protein may, however, have cross reactivity to other antigens, such as LAG-3 proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
The term "human antibody" , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human  germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term "human antibody" , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
The term "recombinant human antibody" , as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or trans-chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below) , (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
The term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen. "
The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.
The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
As used herein, an antibody that "specifically binds human LAG-3" is intended to refer to an antibody that binds to human LAG-3 protein (and possibly a LAG-3 protein from one or more non-human species) but does not substantially bind to non-LAG-3 proteins. Preferably, the antibody binds to a human LAG-3 protein with "high affinity" , namely with a KD of 1 × 10-7 M or less, more preferably 5 × 10-8 M or less, more preferably 3 × 10-8 M or less, more preferably 1 × 10-8 M or less, more preferably 25 × 10-9 M or less or even more preferably 1 × 10-9 M or less.
The term "does not substantially bind" to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a KD of 1 × 10-6 M or more, more preferably 1 × 10-5 M or more, more preferably 1 ×10-4 M or more, more preferably 1 × 10-3 M or more, even more preferably 1 × 10-2 M or more. The term "Kassoc" or "Ka" , as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" or "Kd, " as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD , " as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) . KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a
Figure PCTCN2017074365-appb-000001
system.
The term "high affinity" for an IgG antibody refers to an antibody having a KD of 1 x 10-7 M or less, more preferably 5 x 10-8 M or less, even more preferably 1 x 10-8 M or less, even more preferably 5 x 10-9 M or less, and even more preferably 1 x 10-9 M or less for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10-6 M or less, more preferably 10-7 M or less, even more preferably 10-8 M or less.
The term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
Various aspects of the invention are described in further detail in the following subsections.
Anti-LAG-3 Antibodies Having Particular Functional Properties
The antibodies of the invention are characterized by particular functional features or properties of the antibodies. For example, the antibodies specifically bind to human LAG-3 and may bind to LAG-3 from certain other species, e.g., monkey LAG-3, e.g., cynomolgus monkey, rhesus monkey, but may not substantially bind to LAG-3 from certain other species, e.g., mouse LAG-3. Preferably, an antibody of the invention binds to human LAG-3 with high affinity.
The ability of the antibody to stimulate an immune response, such as an antigen-specific T cell response, can be indicated by, for example, the ability of the antibody to stimulate interleukin-2 (IL-2) or interferon gamma (IFN-γ) production in an antigen-specific T cell response. In certain embodiments, an antibody of the invention binds to human LAG-3 and exhibits an ability to stimulate an antigen-specific T cell response. In other embodiments, an antibody of the invention binds to human LAG-3 but does not exhibit an ability to stimulate an antigen-specific T cell response. Other means by which to evaluate the ability of the antibody to stimulate an immune response include the ability of the antibody to inhibit tumor growth, such as in an in vivo tumor graft model or the ability of the antibody to stimulate an autoimmune response, such as the ability to promote the development of an autoimmune disease in an autoimmune model, such as the ability to promote the development of diabetes in the NOD mouse model.
The binding of an antibody of the invention to LAG-3 can be assessed using one or more techniques well established in the art. For example, in a preferred embodiment, an antibody can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human LAG-3, such as CHO cells that have been transfected to express LAG-3, e.g., human LAG-3, or monkey LAG-3, e.g., rhesus or cynomolgus monkey or mouse LAG-3 on their cell surface. Other suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4+ activated T cells, which express native LAG-3. Additionally, or alternatively, the binding of the antibody, including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays. Still other suitable binding assays include ELISA assays, for example using a recombinant LAG-3 protein. Preferably, an antibody of the invention binds to a LAG-3 protein with a KD of 5 x 10-8 M or less, binds to a LAG-3 protein with a KD of 2 x 10-8 M or less, binds to a LAG-3 protein with a KD of 5 x 10-9 M or less, binds to a LAG-3 protein with a KD of 4 x 10-9 M or less, binds to a LAG-3 protein with a KD of 3 x 10-9 M or less, binds to a LAG-3 protein with a KD of 2 x 10-9 M or less, binds to a LAG-3 protein with a KD of 125 x 10-9 M or less, binds to a LAG-3 protein with a KD of 5 x 10-10 M or less, or binds to a LAG-3 protein with a KD of 1 x 10-10 M or less.
Monoclonal Antibodies
Preferred antibodies of the invention are the human monoclonal antibodies S27, S31, T99, and S119 isolated and structurally characterized as described [Examples 2-8] . The VH amino acid sequences of S27, S31, T99 and S119 are shown in SEQ ID NO: 149, SEQ NO: 150, SEQ ID NO: 158, and SEQ ID NO: 162, respectively. The VL amino acid sequences of S27, S31, T99, and S119 are shown in SEQ ID NO: 198, SEQ NO: 199, SEQ ID NO: 207, and SEQ ID NO: 211, respectively.
Given that each of these antibodies can bind to human LAG-3, the VH and VL sequences can be "mixed and matched" to create other anti-LAG-3 binding molecules of the invention. Preferably, when VH and VL chains are mixed and matched, a VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, preferably a VL sequence from a particular VH/VL pairing is replaced with a structurally similar VL sequence.
Accordingly, in one aspect, this disclosure provides an isolated monoclonal antibody, or antigen binding portion thereof comprising:
(a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 140-SEQ ID NO: 188 and
(b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 189-SEQ ID NO: 237
wherein the antibody specifically binds human LAG-3.
Preferred variable heavy and variable light chain combinations include:
i. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 198；
ii. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 199；
iii. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 207；
iv. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 211.
It is well known in the art that the CDR3 domain, independently from the CDR1and/or CDR2 domain (s) , alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., Brit. J. of Can. 83 (2) : 252-60, 2000； Beiboer et al., J. Mol. Biol. 296: 833-49, 2000； Rader et al., PNAS 95: 8910-15, 1998； Barbas et al., JACS 116: 2161-2, 29914； Barbas et al., PNAS 92: 2529-33, 1995； Ditzel et al., J. Immunol. 157: 739-49, 1996； Berezov et al., BIAJournal 8 (1) : Scientific Review, 2001； Igarashi et al., J. Biochem 117: 452-7, 1995； Bourgeois et al., J. Virol. 72: 807-10, 1998； Levi et al., PNAS 90: 4374-8, 1993； Polymenis and Stoller, J. Immunol. 152: 5218-329, 1994； and Xu and Davis, Immunity 13: 37-45, 2000. See also, U.S. Patent Nos. 6,951,646； 6,914,128； 6,090,382； 6,818,216； 6,156,313； 6,827,925； 5,833,943； 5,762,905 and 5,760,185. Each of these references is hereby incorporated by reference in its entirety.
Accordingly, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from an antibody derived from a human or non-human animal, wherein the monoclonal antibody is capable of specifically binding to human LAG-3. Within certain aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody,  such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to LAG-3. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody (a) are capable of competing for binding with； (b) retain the functional characteristics； (c) bind to the same epitope； and/or (d) have a similar binding affinity as the corresponding parental non-human antibody. Within other aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a human antibody, such as, e.g., a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to human LAG-3. Within other aspects, the present disclosure provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to human LAG-3 and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for LAG-3 to generate a second human antibody that is capable of specifically binding to human LAG-3. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of competing for binding with； (b) retain the functional characteristics； (c) bind to the same epitope； and/or (d) have a similar binding affinity as the corresponding parental first human antibody.
Homologous Antibodies
In yet another embodiment, an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-LAG-3 antibodies of the invention. For example, this disclosure provides an isolated monoclonal antibody, or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80％homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 140-188； (b) the light chain variable region comprises an amino acid sequence that is at least 80％homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 189-237； (c) the antibody specifically binds to human LAG-3； (d) blocks LAG-3 binding to major  histocompatibility complex (MHC) class II molecules； (e) stimulates an immune response； and (f) reverses the inhibitory effect of regulatory T cells on effector cells.
Additionally, or alternatively, the antibody can possess one or more of the following functional properties discussed above, such as high affinity binding to human LAG-3, binding to monkey LAG-3, lack of binding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHC Class II molecules and/or the ability to stimulate antigen-specific T cell responses.
In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody or a chimeric antibody. In other embodiments, the VH and/or VL amino acid sequences can be 85％, 90％, 95％, 96％, 97％, 98％, or 99％homologous to the sequences set forth above. An antibody having VH and VL regions having high (i.e., 80％or greater) homology to the VH and VL regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acids of VH and/or VL amino acid sequences, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth above) using the functional assays described herein.
As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., ％homology ＝ #of identical positions/total #of positions x 100) , taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4: 11-7, 1988) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-53, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
Additionally, or alternatively, the protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against public databases to, e.g., to identify  related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (J. Mol. Biol. 215: 403-10, 1990) . BLAST protein searches can be performed with the XBLAST program, score ＝ 50, word length ＝3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucl. Acid Res. 25 (17) : 3389-402, 1997) . When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are useful.
Engineered and Modified Antibodies
As used herein, the terms “humanized” , “humanization” , and the like, refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (Methods, 36 (1) : 25-34, 2005) and Hou et al. (J. Biochem. 144 (1) : 115-20, 2008) , respectively, to improve various antibody properties, as discussed below.
As used herein, the term "FR" or "framework sequence" refers to any one of FRs 1 to 4. Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present. For example, this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human. Substantially human frameworks are those that have at least 80％sequence identity to a known human germline framework sequence. Preferably, the substantially human frameworks have at least 85％, at least 86％, at least 87％, at least 88％, at least 89％, at least 90％, at least 91％, at least 92％, at least 93％, at least 94％, at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.
Fully human frameworks are those that are identical to a known human germline framework sequence. Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.
The Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences. The individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci. For example, germline light chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39 and IGKV1D-33 and IGKJ1-5 and germline heavy chain FRs can be selected from the group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20, IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59 and IGHV5-51 and IGHJ1-6.
Substantially human FRs are those that have at least 80％sequence identity to a known human germline FR sequence. Preferably, the substantially human frameworks have at least 85％, at least 90％, at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.
CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80％, at least 85％, at least 90％, at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％sequence identity to a CDR sequence disclosed herein. Alternatively, CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein. Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.
Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods Almagro et al. (Front. Biosci., Humanization of antibodies Jan 1 (13) : 1619-33, 2008) . In one approach, the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework. The sequence identity of  the new framework will generally be at least 80％, at least 85％, at least 86％, at least 87％, at least 88％, at least 89％, at least 90％, at least 91％, at least 92％, at least 93％, at least 94％, at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％sequence identical to the sequence of the corresponding framework in the parent antibody compound. In the case of frameworks having fewer than 100 amino acid residues, one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (PNAS 88: 2869, 1991) . Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. (Bioinformatics Feb 1； 31 (3) : 434-5, 2015) and U.S. Patent Nos. 4,816,397, 5,225,539, and 5,693,761； and the method of Winter and co-workers (Jones et al., Nature 321: 522-5, 1996； Riechmann et al., Nature 332: 323-7, 1988； and Verhoeyen et al., Science 239: 1534-6, 1988) .
Humanization began with chimerization, a method developed during the first half of the 1980’s (Morrison et al., PNAS 81: 6851-5, 1984) , consisting of combining the variable (V) domains of murine antibodies with human constant (C) domains to generate molecules with ～70％of human content.
Several different methods can be used to generate humanized antibodies, which are described herein. In one approach, the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new FR will generally be at least 80％, at least 85％, at least 90％, at least 95％, at least 96％, at least 97％, at least 98％, or at least 99％identical to the sequence of the corresponding FR in the parent antibody compound. In the case of FRs having fewer than 100 amino acid residues, one, two, three, four, five, or more amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the FR can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. Bioinformatics. 2015 Feb 1； 31 (3) : 434-435 and U.S. Patents 4,816,397, 5,225,539, and 5,693,761； and the method of  Winter and co-workers (Jones et al. (1986) Nature 321: 522-525； Riechmann et al. (1988) Nature 332: 323-327； and Verhoeyen et al. (1988) Science 239: 1534-1536.
The identification of residues to consider for back-mutation can be carried out as described below. When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the "acceptor FR" ) is replaced by a framework amino acid from a framework of the parent antibody compound (the "donor FR" ) :
i. the amino acid in the human FR of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position；
ii. the position of the amino acid is immediately adjacent to one of the CDRs； or
iii. any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three-dimensional immunoglobulin model.
When each of the amino acids in the human FR of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody compound.
Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.
Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific FRs in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu et al. (1999) J. Mol. Biol. 294: 151-162.
Applying the teachings of the present disclosure, a person skilled in the art can use common techniques, e.g., site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and FR sequences and thereby generate further variable region amino  acid sequences derived from the present sequences. Up to all naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to screen these additional variable region amino acid sequences to identify sequences having the indicated in vivo functions. In this way, further sequences suitable for preparing humanized antibodies and antigen-binding portions thereof in accordance with the present disclosure can be identified. Preferably, amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.
That the functional properties of the antibody compounds generated by introducing the amino acid modifications discussed above conform to those exhibited by the specific molecules disclosed herein can be confirmed by the methods in Examples disclosed herein.
As described above, to circumvent the problem of eliciting human anti-murine antibody (HAMA) response in patients, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients.
To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody-ligand interaction, called “SDR grafting” , has been developed, wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks. This procedure, described in Kashmiri et al. (2005) Methods 36 (1) : 25-34, involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen–antibody complexes of known structures, or by mutational analysis of the antibody-combining site. An alternative approach to humanization involving retention of more CDR residues is based on grafting of the ‘abbreviated’ CDRs, the stretches of CDR residues that include all the SDRs.  Kashmiri et al. also discloses a procedure to assess the reactivity of humanized antibodies to sera from patients who had been administered the murine antibody.
Another strategy for constructing human antibody variants with improved immunogenic properties is disclosed in Hou et al. (2008) J. Biochem. 144 (1) : 115-120. These authors developed a humanized antibody from 4C8, a murine anti-human CD34 monoclonal antibody, by CDR grafting using a molecular model of 4C8 built by computer-assisted homology modelling. Using this molecular model, the authors identified FR residues of potential importance in antigen binding. A humanized version of 4C8 was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the murine antibody FR, together with the murine CDR residues. The resulting humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to murine anti-CD34 antibodies routinely used clinically.
Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated mAbs can contain the set of CDRs from a single murine mAb disclosed herein, or light and heavy chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs. Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a “mix and match” approach to create novel mAbs comprising a mixture of CDRs from the disclosed murine mAbs to achieve new, or improved, therapeutic activities.
Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that "compete" with the molecules disclosed herein are those that bind human LAG-3 at site (s) that are identical to, or overlapping with, the site (s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human LAG-3 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discrete sites on LAG-3, the labeled  compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind. For purposes of the present disclosure, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to LAG-3 by about 50％, about 60％, about 70％, about 80％, about 85％, about 86％, about 87％, about 88％, about 89％, about 90％, about 91％, about 92％, about 93％, about 94％, about 95％, about 96％, about 97％, about 98％, or about 99％. Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Such assays can be made quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor. The capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.
Whether mAbs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods disclosed in the Examples presented below. In various embodiments, competing antibodies for use in the therapeutic methods encompassed herein possess biological activities as described herein in the range of from about 50％to about 100％or about 125％, or more, compared to that of the antibody compounds disclosed herein. In some embodiments, competing antibodies possess about 50％, about 60％, about 70％, about 80％, about 85％, about 90％, about 91％, about 92％, about 93％, about 94％, about 95％, about 96％, about 97％, about 98％, about 99％, or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below.
In addition, or alternative to modifications made within the framework or CDR regions, antibodies of the invention can be engineered to include modifications within the Fc region of  any of the isotypes described, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity (ADCC) . Furthermore, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The mAbs or antigen-binding fragments thereof, or competing antibodies useful in the compositions and methods can be any of the isotypes described herein. Furthermore, any of these isotypes can comprise further amino acid modifications as follows.
The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG1 isotype.
The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life. Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007) . The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al., 2006, Yeung et al., 2009) ； M252Y/S254T/T256E (Dall’A cqua et al., 2006) ； T250Q/M428L (Hinton et al., 2006) ； and M428L/N434S (Zalevsky et al., 2010) .
As opposed to increasing half-life, there are some circumstances where decreased half-life would be desired, such as to reduce the possibility of adverse events associated with high Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) antibodies (Presta 2008) . The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to amino acid modifications I253A (Petkova et al., 2006) ； P257I/N434H, D376V/N434H (Datta-Mannan et al., 2007) ； and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005) .
The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) ,  Antibody-Dependent Cellular Phagocytosis (ADCP) , C1q binding, and altered binding to Fc receptors.
The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al., 2001) ； S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006) ； F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al., 2007) ； G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008) ； K326A/E333A, K326A/E333S and K326W/E333S (Idusogie et al., 2001) ； S267E and S267E/L328F (Smith et al., 2012) ； H268F/S324T, S267E/H268F, S267E/S234T, and S267E/H268F/S324T (Moore et al., 2010) ； S298G/T299A (Sazinsky et al., 2008) ； E382V/M428I (Jung et al., 2010) .
The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al., 1993, Walker et al., 1989) ； L234A/L235A (Xu et al., 2000) ； K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al., 2008) ； C226S/C229S/E233P/L234V/L235A (McEarchern et al., 2007) ； S267E/L328F (Chu et al., 2008) .
The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999) ； E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G, G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S, E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S,  P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R, P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S, P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R, P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S, P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268Q/P271G/A330R, P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto, 2013) .
The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.
The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) , Antibody-Dependent Cellular Phagocytosis (ADCP) , and C1q binding, and altered binding to Fc receptors.
The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to the amino acid modification K326A/E333S (Idusogie et al., 2001) .
The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999) ； E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G, G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S, E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R,  G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R, P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S, P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R, P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S, P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268Q/P271G/A330R, P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto, 2013) .
The Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (CH1 domain) , C232S, C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightle et al., 2010) .
The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.
The human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid (s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC) , Complement-Dependent Cytotoxicity (CDC) , or apoptosis activity.
The human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.
The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.
The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP) .
The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to amino acid modifications F234A/L235A (Alegre et al., 1994) ； S228P, L235E and S228P/L235E (Reddy et al., 2000) .
In still another embodiment, the glycosylation of an antibody is modified. For example, a glycosylated antibody can be made (i.e., the antibody lacks glycosylation) . Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such a glycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
Another modification of the antibodies herein that is contemplated by this disclosure is PEGylation. An antibody can be PEGylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the PEGylation is carried out via an acylation reaction or an alkylation reaction with are active PEG molecule (or an analogous reactive water-soluble polymer) . As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI-CIO) alkoxy-oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to  be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0154316 and EP 0401384.
Characterization of Antibody Binding to Antigen
Antibodies of the invention can be tested for binding to human LAG-3 by, for example, standard ELISA. Anti-LAG-3 human IgG antibodies can be further tested for reactivity with a LAG-3 antigen by Western blotting. The binding specificity of an antibody of the invention can also be determined by monitoring binding of the antibody to cells expressing a LAG-3 protein, e.g., flow cytometry. These methods are known in the art. See, e.g., Harlow and Lane (1988) , cited supra.
Immunoconjugates
Antibodies of this invention can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC) . Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In the ADC, the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADC scan be prepared as described in U.S. Patent Nos. 7,087,600； 6,989,452； and 7,129,261； PCT Publications WO 02/096910； WO 07/038658； WO 07/051081； WO 07/059404； WO 08/083312； and WO 08/103693； U.S. Patent Publications 20060024317； 20060004081； and 20060247295； the disclosures of which are incorporated herein by reference.
Bispecific Molecules
In another aspect, the present disclosure features bispecific molecules comprising an anti-LAG-3 antibody linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules that have three or more specificities. In a preferred embodiment, the bispecific molecule comprises a first binding specificity for LAG-3 and a second binding specificity for a triggering molecule that recruits cytotoxic effector cells that can kill a LAG-3  expressing target cell. Examples of suitable triggering molecules are CD64, CD89, CD16, and CD3. See, e.g., Kufer et al., Trends in Biotech. 22 (5) : 238-44, 2004.
In an embodiment, a bispecific molecule has, in addition to an anti-Fc binding specificity and an anti-LAG-3 binding specificity, a third specificity. The third specificity can be for an anti-enhancement factor (EF) , e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. For example, the anti-enhancement factor can bind a cytotoxic T cell (e.g. via CD2, CD3, CDS, CD28, CD4, CD40, or ICAM-1) , other immune regulatory molecules (e.g. via PD-1, PD-L1, CTLA-4, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, CD47 or CD73) or other immune cell, resulting in an increased immune response against the target cell.
Bispecific molecules can come in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's ) linked by a peptide chain, a so-called Bs (scFv) 2 construct. Intermediate-sized bispecific molecules include two different F (ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al., supra； Cao and Suresh, Bioconjugate Chem. 9 (6) : 635-44, 1988； and van Spriel et al., Immunol. Today 21(8) : 391-7, 2000； and the references cited therein.
Pharmaceutical Compositions
In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody of the present disclosure formulated together with a pharmaceutically acceptable earlier. It may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug. The pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immunostimulatory agent, anti-cancer agent, an anti-viral agent, or a vaccine, such that the anti-LAG-3 antibody enhances the immune response against the vaccine.
The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid  binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed.， Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003) , the disclosure of which is incorporated herein by reference. Preferably, a pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active compound can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
The pharmaceutical compounds of the invention can be in the form of pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01％to about ninety-nine percent of active ingredient, preferably from about 0.1％to about 70％, most preferably from about 1％to about 30％of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated； each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
For administration of the antibody, the dosage ranges from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every 3 to 6 months. Preferred dosage regimens for an anti-LAG-3antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months； (ii) every three weeks； (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/mL and in some methods about 25-300 μg/mL.
A "therapeutically effective dosage" of an anti-LAG-3 antibody of the invention preferably results in a decrease in severity of disease symptoms, an increase infrequency and  duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumor bearing subjects, a "therapeutically effective dosage" preferably inhibits tumor growth by at least about 20％, more preferably by at least about 40％, even more preferably by at least about 60％, and still more preferably by at least about 80％relative to untreated subjects. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Patent Nos. 5,399,163； 5,383,851； 5,312,335； 5,064,413； 4,941,880； 4,790,824； and 4,596,556) ； (2) micro-infusion pumps (U.S. Patent No. 4,487,603) ； (3) transdermal devices (U.S. Patent No. 4,486,194) ； (4) infusion apparati (U.S. Patent Nos. 4,447,233 and 4,447,224) ； and (5) osmotic devices (U.S. Patent Nos. 4,439,196 and 4,475,196) ； the disclosures of which are incorporated herein by reference.
In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic compounds of the invention cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g., U.S. Patent Nos. 4,522,811； 5,374,548； 5,416,016； and 5,399,331； V.V. Ranade, J. Clin. Pharmacol. 29: 685, 1989； Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038； Bloeman et al. (1995) FEBSLett. 357: 140； M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180； Briscoe etal. (1995) Am. J. Physiol. 1233: 134； Schreier et al. (1994) J. Biol. Chern. 269: 9090； Keinanen and Laukkanen (1994) FEBS Lett. 346: 123； and Killion and Fidler (1994) Immunomethods 4: 273.
Uses and Methods of the Invention
The antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, detection of LAG-3 or enhancement of immune response by blockade of LAG-3. In a preferred embodiment, the antibodies of the present invention are human antibodies. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations. Accordingly, in one aspect, the invention provides a method of modifying an immune response in a subject comprising administering to the subject the antibody, or antigen-binding portion thereof, of the invention such that the immune response in the subject is modified. Preferably, the response is enhanced, stimulated or up-regulated.
Preferred subjects include human patients in need of enhancement of an immune response. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., the T-cell mediated immune response) . In a particular embodiment, the methods are particularly suitable for treatment of cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-LAG-3 antibodies can be administered together with an antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor bearing or virus-bearing subject) . When antibodies to LAG-3 are administered together with another agent, the two can be administered in either order or simultaneously.
The invention further provides methods for detecting the presence of humanLAG-3 antigen in a sample, or measuring the amount of human LAG-3 antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to human LAG-3, under conditions that allow for formation of a complex between the antibody or portion thereof and human LAG-3. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of human LAG-3 antigen in the sample. Moreover, the anti-LAG-3 antibodies of the invention can be used to purify human LAG-3 via immunoaffinity purification.
Given the ability of anti-LAG-3 antibodies of the invention to inhibit the binding of LAG-3 to MHC Class II molecules and to stimulate antigen-specific T cell responses, the invention also provides in vitro and in vivo methods of using the antibodies of the invention to stimulate, enhance or upregulate antigen-specific T cell responses. For example, the invention  provides a method of stimulating an antigen-specific T cell response comprising contacting said T cell with the antibody of the invention such that an antigen-specific T cell response is stimulated. Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and/or increase cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 production by the antigen specific T cell is stimulated.
The invention also provides a method of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject comprising administering an antibody of the invention to the subject such that an immune response (e.g., an antigen-specific T cell response) in the subject is stimulated. In a preferred embodiment, the subject is a tumor-bearing subject and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a virus-bearing subject and an immune response against the virus is stimulated.
In another aspect, the invention provides a method for inhibiting growth of tumor cells in a subject comprising administering to the subject an antibody of the invention such that growth of the tumor is inhibited in the subject. In yet another aspect, the invention provides a method of treating viral infection in a subject comprising administering to the subject an antibody of the invention such that the viral infection is treated in the subject.
These and other methods of the invention are discussed in further detail below.
Cancer
Blockade of LAG-3 by antibodies can enhance the immune response to cancerous cells in the patient. In one aspect, the present invention relates to treatment of a subject in vivo using an anti-LAG-3 antibody such that growth of cancerous tumors is inhibited. An anti-LAG-3 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, an anti-LAG-3 antibody can be used in conjunction with other immunogenic agents, standard cancer treatments, or other antibodies, as described below.
Accordingly, in one embodiment, the invention provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-LAG-3 antibody, or antigen-binding portion thereof. Preferably, the antibody is a human anti-LAG-3 antibody (such as any of the human anti-human LAG-3 antibodies  described herein) . Additionally or alternatively, the antibody can be a chimeric or humanized anti-LAG-3 antibody.
Preferred cancers whose growth may be inhibited using the antibodies of the invention include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma) , renal cancer (e.g. clear cell carcinoma) , prostate cancer (e.g., hormone refractory prostate adenocarcinoma) , breast cancer, colon cancer and lung cancer (e.g., non-small cell lung cancer) . Additionally, the invention includes refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the invention. Examples of other cancers that can be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144) .
Optionally, antibodies to LAG-3 can be combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , cells, and cells transfected with genes encoding immune stimulating cytokines (He et al (2004) J. Immunol. 173: 4919-28) . Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE  antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below) .
In humans, some tumors have been shown to be immunogenic such as melanomas. By raising the threshold of T cell activation by LAG-3 blockade, the tumor responses in the host can be activated.
LAG-3 blockade is likely to be more effective when combined with a vaccination protocol. Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62； Logothetis, C., 2000, ASCO Educational Book Spring: 300-302； Khayat, D. 2000, ASCO Educational Book Spring: 414-428； Foon, K. 2000, ASCO Educational Book Spring: 730-738； see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds. ) , 1997, Cancer: Principles and Practice of Oncology, Fifth Edition) . In one of these strategies, a vaccine is prepare dusing autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S. A. 90: 3539-43) .
The study of gene expression and large scale gene expression patterns in various tumors has led to the definition of so called tumor specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7) . In many cases, these tumor specific antigens are differentiation antigens expressed in the tumors and in the cell from which the tumor arose, for example melanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be the targets of tumor specific T cells found in the host. LAG-3 blockade can be used in conjunction with a collection of recombinant proteins and/or peptides expressed in a tumor in order to generate an immune response to these proteins. These proteins are normally viewed by the immune system as self antigens and are therefore tolerant to them. The tumor antigen can include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85％of human cancers and in only a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013) . (These somatic tissues may be protected from immune attack by various means) . Tumor antigen can also be "neo-antigens" expressed in cancer cells because of somatic mutations that alter protein sequence or create fusion proteins  between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome) , or idiotype from B cell tumors.
Other tumor vaccines can include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV) , Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV) . Another form of tumor specific antigen which can be used in conjunction with LAG-3 blockade is purified heat shock proteins (HSP) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from the tumor cells and these HSPs are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot &Srivastava (1995) Science269: 1585-1588； Tamura et al. (1997) Science 278: 117-120) .
Dendritic cells (DC) are potent antigen presenting cells that can be used to prime antigen-specific responses. DC's can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332) . DCs can also be transduced by genetic means to express these tumor antigens as well. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler et al. (2000) Nature Medicine 6: 332-336) . As a method of vaccination, DC immunization can be effectively combined with LAG-3 blockade to activate more potent anti-tumor responses.
LAG-3 blockade can also be combined with standard cancer treatments. LAG-3 blockade can be effectively combined with chemotherapeutic regimes. In these instances, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304) . An example of such a combination is an anti-LAG-3 antibody in combination with decarbazine for the treatment of melanoma. Another example of such a combination is an anti-LAG-3antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of LAG-3 blockade and chemotherapy is that cell death, that is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with LAG-3 blockade through cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with LAG-3 blockade. Inhibition of angiogenesis leads to tumor cell death which may feed tumor antigen into host antigen presentation pathways.
LAG-3 blocking antibodies can also be used in combination with bispecific antibodies that target Fca or Fey receptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243) . Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecific antibodies have been used to target macrophages to sites of tumor. This targeting may more effectively activate tumor specific responses. The T cell arm of these responses would be augmented by the use of LAG-3 blockade. Alternatively, antigen may be delivered directly to DCs by the use of bispecific antibodies which bind to tumor antigen and a dendritic cell specific cell surface marker.
Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins which are expressed by the tumors and which are immunosuppressive. These include among others TGF-β (Kehrl etal. (1986) J. Exp. Med. 163: 1037-1050) , IL-10 (Howard &O'Garra (1992) Immunology Today 13: 198-200) , and Fas ligand (Hahne et al. (1996) Science 4: 1363-1365) . Antibodies to each of these entities can be used in combination with anti-LAG-3 to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
Other antibodies which activate host immune responsiveness can be used in combination with anti-LAG-3. These include molecules on the surface of dendritic cells which activate DC function and antigen presentation. Anti-CD40 antibodies are able to substitute effectively for T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with LAG-3 antibodies (Ito et al. (2000) Immunobiology201 (5) 527-40) . Activating antibodies toT cell costimulatory molecules such as CTLA-4 (e.g., US Patent No. 5,811,097) , OX-40 (Weinberg et al. (2000) Immunol. 164: 2160-2169) , 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997) , and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide for increased levels of T cell activation.
Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. While graft versus host disease is a consequence of this treatment, therapeutic benefit may be obtained from graft vs. tumor responses. LAG-3 blockade can be used to increase the effectiveness of the donor engrafted tumor specific T cells.
There are also several experimental treatment protocols that involve ex vivo activation and expansion of antigen specific T cells and adoptive transfer of these cells into recipients in order to stimulate antigen-specific T cells against tumor (Greenberg &Riddell (1999) Science  285: 546-51) . These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-LAG-3 antibodies can increase the frequency and activity of the adoptively transferred T cells.
Infectious Diseases
Other methods of the invention are used to treat patients that have been exposed to particular toxins or pathogens. Accordingly, another aspect of the invention provides a method of treating an infectious disease in a subject comprising administering to the subject an anti-LAG-3 antibody, or antigen-binding portion thereof, such that the subject is treated for the infectious disease. Preferably, the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) . Additionally or alternatively, the antibody can be a chimeric or humanized antibody.
Similar to its application to tumors as discussed above, antibody mediated LAG-3 blockade can be used alone, or as an adjuvant, in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach can be particularly useful, include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to HIV, Hepatitis (A, B, &C) , Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. LAG-3 blockade is particularly useful against established infections by agents such as HIV that present altered antigens over the course of the infections. These novel epitopes are recognized as foreign at the time of anti-human LAG-3 administration, thus provoking a strong T cell response that is not dampened by negative signals through LAG-3.
Some examples of pathogenic viruses causing infections treatable by methods ofthe invention include HIV, hepatitis (A, B, or C) , herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-11, and CMV, Epstein Barr virus) , adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTL-V virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JCvirus and arboviral encephalitis virus.
Some examples of pathogenic bacteria causing infections treatable by methods ofthe invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas,  legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
Some examples of pathogenic fungi causing infections treatable by methods of the invention include Candida albicans, krusei, glabrata, tropicalis, etc. ) , Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc. ) , Genus Mucorales (mucor, absidia, rhizopus) , Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
Some examples of pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.
In all of the above methods, LAG-3 blockade can be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2) , or bispecific antibody therapy, which provides for enhanced presentation of tumor antigens (see, e.g., Bolliger (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448； Poljak (1994) Structure 2: 1121-1123) .
Autoimmune Reactions
Anti-LAG-3 antibodies may provoke and amplify autoimmune responses. Indeed, induction of anti-tumor responses using tumor cell and peptide vaccines revealsthat many anti-tumor responses involve anti-self reactivities (van Elsas et al. (2001) J. 112 Exp. Med. 194: 481-489； Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987； Hurwitz, (2000) supra； Rosenberg &White (1996) J. Immunother Emphasis Tumor Immunol. 19 (1) : 81-4) . Therefore, it is possible to consider using anti-LAG-3 blockade in conjunction with various self-proteins in order to devise vaccination protocols to efficiently generate immune responses against these self-proteins for disease treatment. For example, Alzheimer's disease involves inappropriate accumulation of Αβ peptide in amyloid deposits in the brain； antibody responses against amyloid are able to clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177) .
Other self-proteins can also be used as targets such as IgE for the treatment of allergy and asthma, and TNFα for rheumatoid arthritis. Finally, antibody responses to various hormones may be induced by the use of anti-LAG-3 antibody. Neutralizing antibody responses to  reproductive hormones can be used for contraception. Neutralizing antibody response to hormones and other soluble factors that are required for the growth of particular tumors can also be considered as possible vaccination targets.
Analogous methods as described above for the use of anti-LAG-3 antibody can be used for induction of therapeutic autoimmune responses to treat patients having an inappropriate accumulation of other self-antigens, such as amyloid deposits, including Αβ in Alzheimer's disease, cytokines such as TNFα, and IgE.
Vaccines
Anti-LAG-3 antibodies can be used to stimulate antigen-specific immune responses by co-administration of an anti-LAG-3 antibody with an antigen of interest (e.g., a vaccine) . Accordingly, in another aspect the invention provides a method of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) the antigen； and (ii) an anti-LAG-3 antibody, or antigen-binding portion thereof, such that an immune response to the antigen in the subject is enhanced. Preferably, the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) . Additionally or alternatively, the antibody can be a chimeric or humanized antibody. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria or other pathogens described above.
Suitable routes of administering the antibody compositions (e.g., human monoclonal antibodies, multi-specific and bispecific molecules and immunoconjugates) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous) . Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
As previously described, human anti-LAG-3 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immuno-complex) or can be administered separate from the agent. In the latter case (separate administration) , the antibody can be administered before, after or concurrently with the agent or  can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin) , cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/mL dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/mL dose once every 21 days. Co-administration of the human anti-LAG-3 antibodies, or antigen binding fragments thereof, of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.
Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., human antibodies, bispecific or multi-specific molecules, or immunoconjugates) and instructions for use. The kit can further contain at least one additional reagent, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope inLAG-3 antigen distinct from the first human antibody) . Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
Nervous System Disorders
Other methods of the invention are used to treat patients that have a progressive disorder of the nervous system that affects movement. In one embodiment, the progressive disorder of the nervous system that affects movement is Parkinson’s disease. Accordingly, another aspect of the invention provides a method of treating Parkinson’s disease in a subject comprising administering to the subject an anti-LAG-3 antibody, or antigen-binding portion thereof, such that the subject is treated for Parkinson’s disease. Preferably, the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein) . Additionally or alternatively, the antibody can be a chimeric or humanized antibody.
In addition to immune system organ e.g. thymus and spleen, LAG3 is enriched in the brain as well (C.J. Workman (2002) ， Eur. J. Immunol. 32, 2255–2263) . Immunoblot analysis  indicates that LAG3 is expressed predominantly in neurons. According to the Allen Brain Atlas, LAG3 is localized to neurons throughout the central nervous system (CNS) , including DA neurons. X. Mao et al., (Science 353, aah3374) reported that LAG3 preferentially binds α-synuclein (α-syn) misfolded preformed fibrils (PFF) with high affinity mainly through its D1 domain (29-167AA) . In addition, deletion of the D2 (168-252AA) , D3 (265-343AA) , or intracellular domain (ICD, 472-525AA) substantially weakens binding of LAG3 to α-syn PFF, X. Mao et al have shown that α-syn PFF binding to LAG3 initiated a-syn PFF endocytosis, transmission, and toxicity. Emerging evidence indicates that the pathogenesis of Parkinson’s disease (PD) may be due to cell-to-cell transmission of misfolded α-syn PFF. Parkinson’s disease (PD) is the second most common neurodegenerative disorder and leads to slowness of movement, tremor, rigidity, and, in the later stages of PD, cognitive impairment. Pathologically, PD is characterized by the accumulation of a-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system, with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD. Anti-LAG3 antibody specifically bind to D1 or D2 domain can reduce a-syn PFF toxicity and cell-to-cell transmission, suggesting its potential for PD therapy. As shown in the Example 1, our antibody can specifically bind to D1 or D2 domain of LAG3 protein. Therefore, there antibody can be used for the PD therapy.
Combination Therapy
In another aspect, the invention provides methods of combination therapy in which an anti-LAG-3 antibody is co-administered with one or more additional antibodies that are effective in stimulating immune responses to thereby further enhance, stimulate or upregulate immune responses in a subject. For example, the invention provides a method for stimulating an immune response in a subject comprising administering to the subject an anti-LAG-3 antibody and one or more additional immunostimulatory antibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody, such that an immune response is stimulated in the subject, for example to inhibit tumor growth or to stimulate an anti-viral response. In one embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-1 antibody. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-L1 antibody. In yet another embodiment, the subject is administered ananti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, the anti-LAG-3 antibody is a human antibody, such  as an antibody of the disclosure. Alternatively, theanti-LAG-3 antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse anti-LAG-3 mAb) . In another embodiment, the at least oneadditional immunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse anti-PD-1, anti-PD-L1 and/or anti-CTLA-4 antibody) .
In one embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a CTLA-4 antibody to a subject. In further embodiments, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-CTLA-4 antibody is human sequence monoclonal antibody 10D1 (described in PCT Publication WO 01114424) and the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein. Other anti-CTLA-4 antibodies encompassed by the methods of the present invention include, for example, those disclosed in: WO98/42752； WO 00/37504； U.S. Patent No. 6,207,156； Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95 (17) : 10067-10071； Camacho et al. (2004) J. Clin. Oncology 22 (145) : Abstract No. 2505 (antibody CP-675206) ； and Mokyr et al. (1998) Cancer Res. 58: 5301-5304. In certain embodiments, the anti-CTLA-4 antibody binds to human CTLA-4 with a KD of 5 x 10-8 M or less, binds to human CTLA-4 with a KD of 1 x 10-8 M or less, binds to human CTLA-4 with a KD of 5 x 10-9 M or less, or binds to human CTLA-4 with a KD of between 1 x 10-8 M and 1 x 10-10 M or less.
In one embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a PD-1 antibody to a subject. In further embodiments, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease  with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-1 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-PD-1 antibody is a human sequence monoclonal antibody nd the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein. Examples of human sequence antiPD-1 antibodies include 17D8, 2D3, 4H1, 5C4 and 4A11, which are described in PCT Publication WO 061121168. In certain embodiments, the anti-PD-1 antibody binds to human PD-1 with a KD of 5 x 10-8 M or less, binds to human PD-1 with a KD of 1 x 10-8 M or less, binds to human PD-1 with a KD of 5 x 10-9 M or less, or binds to human PD-1with a KD of between 1 X 10-8 M and 1 X 10-10 M or less.
In one embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) , comprising administering a LAG-3 antibody and a PD-L1 antibody to a subject. In further embodiments, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In another embodiment, the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose ofanti-PD-L1 antibody to a subject. In certain embodiments, the subject is human. In certain embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody and the anti-LAG-3 antibody is human sequence monoclonal antibody, such as S27, S31, T99, or S119 as described herein. Examples of human sequence anti-PD-L1 antibodies include 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7and 13G4, which are described in PCT Publication WO 07/005874. In certain embodiments, the anti-PD-L1 antibody binds to human PD-L1 with a KD of 5 x 10-8 M or less, binds to human PD-L1 with a KD of 1 x 10-8 M or less, binds to human PD-L1with a KD of 5 x 10-9 M or less, or binds to human PD-L1 with a KD of between 1 x 10-8 M and 1 x 10-10 M or less.
Blockade of LAG-3 and one or more second target antigens such as CTLA-4 and/or PD-1 and/or PD-L1 by antibodies can enhance the immune response to cancerous cells in the patient. Cancers whose growth may be inhibited using the antibodies of the instant disclosure include cancers typically responsive to immunotherapy. Representative examples of cancers for treatment with the combination therapy of the instant disclosure include those cancers specifically listed above in the discussion of monotherapy with anti-LAG-3 antibodies.
In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-CTLA-4 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-CTLA-4 antibody second. Additionally or alternatively, an anti-PD-1 antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-PD-1 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-PD-1 antibody second. Additionally or alternatively, an anti-PD-L1 antibody and an anti-LAG-3 antibody can be administered sequentially, such as anti-PD-L1 antibody being administered first and anti-LAG-3 antibody second, or anti-LAG-3 antibody being administered first and anti-PD-Ll antibody second.
Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof. For example, the first administration of a combination anti-CTLA-4 antibody and anti-LAG-3antibody can be concurrent, the second administration can be sequential with anti-CTLA-4 first and anti-LAG-3 second, and the third administration can be sequential with anti-LAG-3 first and anti-CTLA-4 second, etc. Additionally or alternatively, the first administration of a combination anti-PD-1 antibody and anti-LAG-3 antibody can be concurrent, the second administration can be sequential with anti-PD-1 first and anti-LAG-3 second, and the third administration can be sequential with anti-LAG-3 first andanti-PD-1 second, etc. Additionally or alternatively, the first administration of a combination anti-PD-Ll antibody and anti-LAG-3 antibody can be concurrent, the second administration can be sequential with anti-PD-Ll first and anti-LAG-3 second, and the third administration can be sequential with anti-LAG-3 first and anti-PD-Ll second, etc. Another representative dosing scheme can involve a first administration that is sequential with anti-LAG-3 first and anti-CTLA-4 (and/or anti-PD-1 and/or anti-PD-Ll) second, and subsequent administrations may be concurrent.
Optionally, the combination of anti-LAG-3 and one or more additional antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-Ll antibodies) can be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , cells, and cells transfected with genes encoding immune stimulating cytokines (He et al. (2004) J. Immunol. 173: 4919-28) . Non-limiting examples of tumor vaccines that can beused include peptides of melanoma antigens, such as peptides of gpl00, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. A combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-Ll blockade can be further combined with a vaccination protocol, such as any of the vaccination protocols discussed in detail above with respect to monotherapy with anti-LAG-3 antibodies.
A combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can also be further combined with standard cancer treatments. For example, a combined LAG-3and CTLA-4 and/or PD-1 and/or PD-L1 blockade can be effectively combined with chemotherapeutic regimes. In these instances, it is possible to reduce the dose of other chemotherapeutic reagent administered with the combination of the instant disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304) . An example of such a combination is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combination with decarbazine for the treatment of melanoma. Another example is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade with chemotherapy is that cell death, which is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade through cell death include radiation, surgery, or hormone deprivation. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade. Inhibition of angiogenesis leads to tumor cell death, which can be a source of tumor antigen fed into host antigen presentation pathways.
A combination of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blocking antibodies can also be used in combination with bispecific antibodies that target Fcα or Fcγ receptor- expressing effector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5, 922, 845 and 5, 837, 243) . Bispecific antibodies can be used to target two separate antigens. The T cell arm of these responses would be augmented by the use of a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade. In another example, a combination of anti-LAG-3 and anti-CTLA-4 and/or antiPD-1 antibodies and/or anti-PD-L1 antibodies can be used in conjunction with anti-neoplastic antibodies, such as
Figure PCTCN2017074365-appb-000002
 (rituximab) , 
Figure PCTCN2017074365-appb-000003
 (trastuzumab) , 
Figure PCTCN2017074365-appb-000004
(tositumomab) , 
Figure PCTCN2017074365-appb-000005
 (ibritumomab) , 
Figure PCTCN2017074365-appb-000006
 (alemtuzumab) , 
Figure PCTCN2017074365-appb-000007
(eprtuzumab) , 
Figure PCTCN2017074365-appb-000008
 (bevacizumab) , and
Figure PCTCN2017074365-appb-000009
 (erlotinib) , and the like. By wayof example and not wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can lead to cancer cell death (e.g., tumor cells) which would potentiate an immune response mediated by CTLA-4, PD-1, PD-L1 or LAG-3. In an exemplary embodiment, a treatment of a hyperproliferative disease (e.g., a cancer tumor) can include an anti-cancer antibody in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies, concurrently or sequentially or any combination thereof, which can potentiate an anti-tumor immune responses by the host.
Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins, which are expressed by the tumors and which are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050) , IL-10 (Howard &O'Garra (1992) Immunology Today 13: 198-200) , and Fas ligand (Hahne et al. (1996) Science 27 4: 1363-1365) . In another example, antibodies to each of these entities can be further combined with an anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibody combination to counteract the effects of immunosuppressive agents and favor anti-tumor immune responses by the host.
Other antibodies that can be used to activate host immune responsiveness can be further used in combination with an anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1and/or anti-PD-L1 antibody combination. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies (Ridge et al., supra) can be used in conjunction with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 combination (Ito et al., supra) . Other activating antibodies to T cell co stimulatory molecules Weinberg et al., supra, Melero et al. supra, Hutloff et al., supra) may also provide for increased levels of T cell activation.
As discussed above, bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. A combined LAG-3 and CTLA-4 and/orPD-1 and/or PD-L1 blockade can be used to increase the effectiveness of the donor engrafted tumor specific T cells.
Several experimental treatment protocols involve ex vivo activation and expansion of antigen specific T cells and adoptive transfer of these cells into recipients in order to antigen-specific T cells against tumor (Greenberg &Riddell, supra) . These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1and/or anti-PD-L1 antibodies can be expected to increase the frequency and activity of the adoptively transferred T cells.
In certain embodiments, the present invention provides a method for altering an adverse event associated with treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of anti-CTLA-4 and/or anti-PD-1and/or anti-PD-L1 antibody to a subject. For example, the methods of the present invention provide for a method of reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to the patient. Because any patient who will receive an immunostimulatory therapeutic antibody is at risk for developing colitis or diarrhea induced by such an antibody, this entire patient population is suitable for therapy according to the methods of the present invention. Although steroids have been administered to treat inflammatory bowel disease (IBD) and prevent exacerbations of IBD, they have not been used to prevent (decrease the incidence of) IBD in patients who have not been diagnosed with IBD. The significant side effects associated with steroids, even non-absorbable steroids, have discouraged prophylactic use.
In further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1 and/orPD-L1 blockade (i.e., immunostimulatory therapeutic antibodies anti-LAG-3 and antiCTLA-4 and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies) can be further combined with the use of any non-absorbable steroid. As used herein, a "nonabsorbable steroid" is a glucocorticoid that exhibits extensive first pass metabolism such that, following metabolism in the liver, the bioavailability of the steroid is low, i.e., less than about 20％. In one embodiment of the invention, the non-absorbable steroid isbudesonide. Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration. ENTOCORT
Figure PCTCN2017074365-appb-000010
 (Astra-Zeneca) is a pH-and time-dependent oral formulation  of budesonide developed to optimize drug delivery to the ileum and throughout the colon. ENTOCORT
Figure PCTCN2017074365-appb-000011
is approved in the U.S. for the treatment of mild to moderate Crohn's disease involving theileum and/or ascending colon. The usual oral dosage of ENTOCORT
Figure PCTCN2017074365-appb-000012
for thetreatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT
Figure PCTCN2017074365-appb-000013
is released in the intestines before being absorbed and retained in the gut mucosa. Once it passes through the gut mucosa target tissue, ENTOCORT
Figure PCTCN2017074365-appb-000014
is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, the bioavailability is low (about 10％) . The low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less extensive first-pass metabolism. Budesonide results in fewer adverse effects, including less hypothalamic-pituitary suppression, than systemically-acting corticosteroids. However, chronic administration of ENTOCORT
Figure PCTCN2017074365-appb-000015
can result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR 58th ed.2004； 608-610.
In still further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodies anti-LAG-3 andanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies) in conjunction with a nonabsorbable steroid can be further combined with a salicylate. Salicylates include 5-ASAagents such as, for example: sulfasalazine (
Figure PCTCN2017074365-appb-000016
Pharmacia &UpJohn) ； olsalazine (
Figure PCTCN2017074365-appb-000017
Pharmacia &UpJohn) ； balsalazide (
Figure PCTCN2017074365-appb-000018
Salix Pharmaceuticals, Inc. ) ； and mesalamine (
Figure PCTCN2017074365-appb-000019
Procter &Gamble Pharmaceuticals； 
Figure PCTCN2017074365-appb-000020
Shire US； 
Figure PCTCN2017074365-appb-000021
Axcan Scandipharm, Inc.； ROW
Figure PCTCN2017074365-appb-000022
Solvay) .
In accordance with the methods of the present invention, a salicylate administered in combination with anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 antibodies and a non-absorbable steroid can include any overlapping or sequential administration of the salicylate and the non-absorbable steroid for the purpose of decreasing the incidence of colitis induced by the immunostimulatory antibodies. Thus, for example, methods for reducing the incidence of colitis induced by the immunostimulatory antibodies according to the present invention encompass administering a salicylate and a non-absorbable concurrently or sequentially (e.g., a salicylate is administered 6 hours after a non-absorbable steroid) , or any combination thereof. Further, according to the present invention, a salicylate and a non-absorbable steroid can be administered by the same route (e.g., both are administered orally) or  by different routes (e.g., a salicylate is administered orally and a non-absorbable steroid is administered rectally) , which may differ from the route (s) used to administer the anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 antibodies.
The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, GenBank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. In particular, the disclosures of PCT publications WO 09/045957, WO 09/073533, WO 09/073546, and WO 09/054863 are expressly incorporated herein by reference.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Example 1
Screening of full human monoclonal antibodies against LAG-3.
Anti-LAG3 human monoclonal antibodies (α-LAG-3 mAbs) were generated by screening full human Fab phage-display libraries. Wildtype LAG-3-ECD-huFc fragments can bind to Daudi cells while D1-D2 truncated LAG-3-ECD-huFc fragments fail to bind Daudi cells. Consequently, the D1-D2 domains are critical for LAG-3 function.
Antigens for phage-display library-panning. LAG-3 is a single-pass type I membrane protein which belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains (ECD) : domain (D) 1, D2, D3 and D4. A recombinant human LAG-3-ECD-human IgG1 (LAG-3-huFc) fusion protein or a human D1-D2 truncated LAG-3-ECD-human IgG1 (ΔD1D2-LAG-3-huFc) fusion protein were expressed in a 293T cell system.
Phage library. Ig gene segments in mammals are arranged in groups of variable (V) , diversity (D) , joining (J) , and constant (C) exons. The human Fab phage library were construed using the phage vectors, which consists of: 1) all human variable kappa (VK) repertoires； and 2) the VH of VH3-23 and VH1-69 germline genes, respectively, with genetically randomized CDR3 regions from healthy human subjects.
Antigen screening and generation. To select the D1-D2 domain-specific phage binders, the phage libraries were subjected to antigen-based panning.
I) Phage library solution panning against LAG-3.
293F cells were transfected with a plasmid containing a D1-D2 deleted LAG-3 (ΔD1D2-LAG-3) sequence with a FLAG-tag at the N-terminus. At 3 days post-transfection, the ΔD1D2-LAG-3 293F cells were used for phage library screening. The phage libraries were performed the sequential negative screenings: streptavidin beads, △D1D2-LAG-3 transfected 293F cells and biotin-labeled-human IgG1Fc protein. The resulting library was then incubated with biotinylated LAG-3-huFc LAG-3 for 2 hrs under motion, followed by incubation with 100μL of casein blocked streptavidin-magnetic beads for 15 min. Unbound phages were removed by washing with PBS 5-20 times. The bound phages were then eluted with freshly prepared 100mM triethylamine (TEA) and neutralized with the addition of Tris-HCl buffer. The resulting phages were labeled as the Output-1 phage libraries. Output-1 phage libraries were subjected to the same screening as described above to generate the Output-2 and subsequent Output-3 phage libraries. Three rounds of phage library screening were performed in total.
II) Phage library immunotube panning against LAG-3.
The phage libraries were performed the sequential negative screenings: casein-coated immunotubes, ΔD1D2-LAG-3 transfected 293F cells and human IgG1Fc protein. The resulting library was then incubated in LAG3-huFc-coated immunotubes for 2 hrs under motion. Unbound phages were removed by washing with PBST 5-20 times. Similar with cell based panning, three rounds of phage library screening were performed in total.
Output-3 phage libraries were diluted and plated to grow at 37℃ for 8 hrs and captured by anti-kappa antibody-coated filters overnight at 22℃. Biotinylated LAG-3-huFc (50nM) and NeutrAvidin-AP conjugate were applied to the filter to detect antigen binding anti-LAG3 phages. Positive phage plaques were picked and eluted into 100 μL of phage elution buffer. About 10-15 μL of eluted phages were then used to infect 1 mL of XL1-Blue competent cells to make a high-titer (HT) phage for phage single point ELISA (SPE) (ELISA immobilized substrate coated with 50 nM of each protein tested) . 1x1010 plaque forming units (pfus) of each phage hit was used for SPE confirmation. The positive clones picked from the filter lift were then tested for LAG-3 antigen binding with LAG-3-huFc and ΔD1D2-LAG-3-huFc. The D1-D2 specific binders were amplified from antigen positive phages by PCR and sequenced. Ig light chain V genes (VL) and VH sequences were analyzed to identify unique sequences and determine sequence diversity.
VL and VH gene sequences of all hits were cloned into expression vectors pFUSE2ss-CLIg-hk (light chain, InvivoGen Cat No. pfuse2ss-hclk) and pFUSEss-CHIg-hG1 (heavy chain, InvivoGen Cat No. pfusess-hchg1) . The antibodies were expressed in HEK293 cells and purified using Protein A PLUS-Agarose.
Example 2
The binding of human anti-LAG3 antibodies to LAG3 protein derived from various species.
To evaluate the capability of the anti-LAG-3 antibodies to bind to human, rat, and mouse LAG3 the antibodies identified in Example 1 were evaluated for their binding property through ELISA. The human, rat and mouse LAG3ECD-Fc protein were coated to ELISA plate at 1μg/ml with 100μl/well. Antibodies from Example 1 were serially diluted with ELISA diluent buffer. To assess binding, LAG-3 antibodies at various concentrations 10 μg/ml, 3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123 μg/ml, 0.041 μg/ml, 0.014 μg/ml, 0.005 μg/ml, 0.0015 μg/ml and 0.0005 μg/ml) were then added to LAG3 antigen coated plate for 1.5hr RT. The resulting plates were washed and then labeled with anti-human IgG (Fab) -HRP antibody. The S31 can only bind  to human LAG3. The S27 and T99 can bind to human LAG3 and rat/mouse LAG3 with lower potency. The S119 antibody can bind to human, rat and mouse LAG3 at high potency (FIG. 2) .
Example 3
The binding of human anti-LAG3 antibodies to cell surface LAG-3 antigen on activated human primary CD4+ T cells.
LAG-3 is expressed on activated or exhausted T cells. CD4+ T cells were isolated using CD4 magnetic beads. The purified human CD4+ T cells were stimulated with
Figure PCTCN2017074365-appb-000023
Human T-Activator CD3/CD28 for 72 hrs. Antibodies from Example 1 were serially diluted with FACS buffer. To assess binding, LAG-3 antibodies at various concentrations (10 μg/ml, 3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123 μg/ml, 0.041 μg/ml, 0.014 μg/ml and 0.005 μg/ml) were then added to the activated human CD4 T cells in the presence of mouse anti-human LAG3 PE antibody (eBioscience, clone: 3DS223H) for 30 min on ice. The labeled cells were washed with FACS buffer and subsequently labeled with APC-conjugated anti-human IgG antibodies for 30 min on ice. The resulting cells were washed once with FACS buffer. Labeled cells were evaluated for fluorescence intensity by flow cytometry in a BD FACSCaliburTM. As shown in FIG. 3, the S27, S31, T99 and S119 can dose-dependent binding to LAG3 expressed on the activated human CD4+ T cells.
Example 4
Anti-LAG-3 antibody inhibition of soluble LAG-3 (sLAG) binding to MHC class II receptor.
To evaluate the ability of anti-LAG-3 antibodies to block the binding of sLAG-3 to MHC class II receptor, an in vitro binding assay was designed using biotin-labeled LAG-3-ECD-huFcfusion proteins and Raji cells expressing MHC class II receptor. Antibodies from Example 1 were serially diluted from 20μg/mL with FACS buffer and pre-incubated with 6 μg/mL of biotin-LAG-3-ECD-huFcc for 30 min at room temperature. The antibody mixture was then added to FcR blocked Raji cells and incubated for 30 min on ice. Cells were then washed with FACS buffer and subsequently stained with streptavidin PE for 30 min on ice and subsequently washed once with FACS buffer. Labeled cells were evaluated for fluorescence intensity by flow cytometry in a BD FACSCaliburTM. As shown in FIG. 4, the S27, S31, S119 and T99 antibodies can dose dependently inhibit the binding of LAG3 to its receptor MHC class II molecules.
Example 5
Stimulation of IL-2 production in peripheral blood mononuclear cells (PBMCs) by anti-LAG-3 antibodies.
Staphylococcal enterotoxin B (SEB) is a superantigen that simultaneously bindings to MHC class II antigens and T cell receptors (TCRs) , bringing them together in such a way as to induce T cell proliferation and cytokine production. 2 × 105 PBMCs were stimulated with SEB in the presence of the antibodies from Example 1 at various concentrations starting from 20μg/ml at 1: 3 serious dilution for 6 doses. Three days later, IL-2 concentration in the culture supernatant was evaluated by ELISA. As shown in FIG. 5, and similar with PD-1 antibody, the anti-LAG3 antibodies: S24, S27, S31, S87, S119, T99 and S20 can dose dependently enhance the IL-2 production as compared with SEB stimulation only.
Example 6
Reversing the inhibition of regulatory T cells (Tregs) on effector T cells (Teffs) using anti- LAG-3  antibodies.
LAG-3 is highly expressed on Tregs (CD4+CD25hi) and mediates their suppressive function (Journal of Immunology 184: 6545-51, 2010) . To evaluate the ability of anti-LAG-3 antibodies on reversing the suppressive effect of Tregs on effector T cells (CD4+CD25-CD127hi) , the antibodies of Example 1 were used in an in vitro suppression assay. First, Tregs (CD4+CD25hiCD127low) and Teffs (CD4+CD25-CD127hi) were FACS-sorted by using a BD FACSAria II system. Teffs were then labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with Tregs at a 1: 1 ratio in the presence of plate bound anti-CD3 antibodies and mitomycin C-treated antigen presenting cells. Anti-LAG-3 antibodies were next added to the cell culture and Teffs cell proliferation were tested 5 days later. The results in FIG. 6, indicate that when Tregs were co-cultured with effector T cells, effector T cell proliferation and cytokine production was inhibited. S119 and T99 can reverse the inhibition of Teffs by Tregs.
Example 7
Synergistic effect of anti-LAG3 and PD-1 antibody combo treatment.
Staphylococcal enterotoxin B (SEB) is a superantigen that stimulate the human immune response. PD-1 blocking antibody can enhance the SEB stimulated IL-2 production. As shown in Example 5, anti-LAG3 antibodies can also highlight the SEB mediated IL-2 production. To explore the effect of an anti-LAG3 antibody in combination with PD-1 antibody, we investigated the effect of anti-LAG3 antibody on SEB stimulation in the presence of suboptimal PD-1  stimulation. In the presence of 0.1μg/ml PD-1 antibody, the serious diluted anti-LAG3 antibodies were added to the SEB culture. IL-2 production were evaluated 72hr later. The results in FIG. 7 indicate that anti-LAG-3 antibodies can enhance the SEB stimulated T cell response in a dose-dependent manner in the presence of suboptimal PD-1 treatment, suggesting that anti-LAG3 and anti-PD-1 combo treatment have synergistic effect.
Example 8
Anti-LAG-3 antibodies enhance human T cell response in the presence of PD-L1 antibody.
To evaluate the effect of anti-LAG-3 antibodies in combination with PD-L1 antibody, the response of human T cells assessed in a mixed lymphocyte reaction setting. Human DCs were differentiated from CD14+ monocytes in the presence of GM-CSF and IL-4 for 7 days. CD4+ T cells isolated from another donor were then co-cultured with the DCs and serial dilutions of anti-LAG-3 antibodies and PD-L1 blocking antibody. At day 2 post-inoculation, the culture supernatant was assayed for IL-2 production. The results in FIG. 8 indicate that anti-LAG-3 antibodies can significantly promote IL-2 production in conjunction with a PD-L1 antibody.
Example 9
LAG-3 antibody synergy with PD-1/PD-L1 blockade.
Cells from the human lung adenocarcinoma cell line HCC827 will be grafted into NOD scid gamma (NSG) mice. NSG mice are NOD scid gamma deficient and the most immunodeficient mice making them ideal recipients for human tumor cell and PBMC grafting. 10 days post-graft, human PBMCs will be transplanted into the tumor-bearing mice. Approximately 20 days post-graft, once the tumor volume has reached 100-150mm3, PD-1/PD-L1 antibodies (2mg/kg) alone, or PD-L1 and LAG-3 (10mg/kg for each LAG3 antibody) antibodies together, will be administered to the mice every other day. Tumor volume will be monitored every other day in conjunction with antibody administration. The 2mg/kg PD-L1/PD-1 antibody will show minimal effect on tumor volume. The LAG3 combo will significantly shrink the tumor volume.
Example 10
LAG-3 antibody BIACORE Analysis
The binding of the S20, S24, S27, S31, S87, S119, S120, S128, S136, S161 and T99 antibodies to recombinant his-taq human LAG3-ECD protein was examined by BIAcore T200 using a capture method. The anti-LAG3 antibodies were captured using anti-human Fc antibody.  The anti-human Fc antibody was coated on chip. The serious concentration of his-taq human LAG3-ECD protein (0-4nM) were injected over capture antibodies at the flow rate of 30 μl/min. The dissociation phase were 900s or 550s. The results are shown in Table 1 below. The Biacore results for the anti-LAG3 antibodies have shown that these anti-LAG3 antibodies are high affinity binder to human LAG3.
Table 1.
  Ka (M-1s-1) kd (s-1) KD (M)
S20 1.65E+05 7.33E-06 4.43E-11
S24 1.79E+06 1.20E-02 6.73E-09
S27 7.04E+06 1.10E-04 1.56E-11
S31 2.08E+06 6.25E-05 3.00E-11
S87 9.28E+05 2.33E-06 2.51E-12
S119 2.17E+07 1.49E-04 6.87E-12
S120 1.40E+06 2.64E-03 1.88E-09
S128 1.00E+06 8.17E-04 8.15E-10
S136 7.98E+05 8.27E-05 1.04E-10
S161 6.20E+05 5.53E-04 8.92E-10
T99 7.62E+06 1.70E-04 2.24E-11

Claims (39)

  1. A monoclonal antibody, or an antigen binding fragment thereof, wherein the antibody:
    i. binds human LAG-3,
    ii. blocks LAG-3 binding to major histocompatibility (MHC) class II molecules；
    iii. stimulates an immune response； and
    iv. reverses the inhibitory effect of regulatory T cells on effector T cells.
  2. The monoclonal antibody, or antigen binding fragment thereof of claim 1, which is a chimeric or humanized antibody.
  3. The monoclonal antibody, or antigen binding fragment thereof according to any one of the preceding claims, wherein the monoclonal antibody, or antigen binding fragment thereof cross-reacts with one or more species homologs of LAG-3.
  4. The monoclonal antibody, or antigen binding fragment thereof according to any one of the preceding claims, wherein the antibody stimulates interleukin-2 (IL-2) production in an antigen-specific T cell response.
  5. The monoclonal antibody, or antigen binding fragment thereof according to any one of the preceding claims, wherein the antibody stimulates interferon gamma (IFN-γ) production in an antigen-specific T cell response.
  6. The monoclonal antibody, or antigen binding fragment thereof according to any one of the preceding claims, wherein the antibody stimulates an anti-tumor immune response.
  7. The monoclonal antibody, or antigen binding fragment thereof according to any one of the preceding claims, wherein the antibody binds to human LAG-3 with a Kd of 1 × 10-8 or less.
  8. The monoclonal antibody, or antigen binding fragment thereof of according to any one of the preceding claims, wherein the antibody binds to human LAG-3 with a Kd of 1 × 10-10 or less.
  9. The monoclonal antibody or antigen binding fragment thereof, according to any one of the preceding claims, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3,
    wherein said variable chain CDR1 comprises the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2；
    wherein said variable heavy chain CDR2 comprises the amino acid sequence SEQ ID NO: 3 or SEQ NO: 4； and
    wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45.
  10. The monoclonal antibody or antigen binding fragment thereof, of claim 9, further comprising a light chain variable domain comprising a variable light chain CDR1, variable light chain CDR2, and a variable light chain CDR3, wherein said variable chain CDR1 comprises the amino acid sequence SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID  NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or SEQ ID NO: 115；
    wherein said variable light chain CDR2 comprises the amino acid sequence SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 98； and
    wherein said variable light chain CDR3 comprises the amino acid sequence SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139..
  11. The monoclonal antibody or antigen binding fragment thereof, of claims 9-10, comprising a combination of variable heavy chain CDR1 (HCDR1) , variable heavy chain CDR2 (HCDR2) , and a variable heavy chain CDR3 (HCDR3) , variable light chain CDR1 (LCDR1) , variable light chain CDR2 (LCDR2) , and a variable light chain CDR3 (LCDR3) , wherein the combination is selected from the group consisting of:
    i. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 5, LCDR1 comprising SEQ ID NO: 46, LCDR2 comprising SEQ ID NO: 81, LCDR3 comprising SEQ ID NO: 104；
    ii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 6, LCDR1 comprising SEQ ID NO: 47, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105；
    iii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 7, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106；
    iv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 8, LCDR1 comprising SEQ ID NO: 49, LCDR2 comprising SEQ ID NO: 83, LCDR3 comprising SEQ ID NO: 105；
    v. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 9, LCDR1 comprising SEQ ID NO: 50, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 106；
    vi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 107；
    vii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 11, LCDR1 comprising SEQ ID NO: 51, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 108；
    viii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 109；
    ix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 13, LCDR1 comprising SEQ ID NO: 52, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 104；
    x. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 14, LCDR1 comprising SEQ ID NO: 53, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 110；
    xi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
    xii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 16, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 105；
    xiii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 17, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112；
    xiv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 18, LCDR1 comprising SEQ ID NO: 56, LCDR2 comprising SEQ ID NO: 87, LCDR3 comprising SEQ ID NO: 113；
    xv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
    xvi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 19, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 112；
    xvii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 20, LCDR1 comprising SEQ ID NO: 57, LCDR2 comprising SEQ ID NO: 98, LCDR3 comprising SEQ ID NO: 114；
    xviii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 115；
    xix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 22, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 116；
    xx. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 23, LCDR1 comprising SEQ ID NO: 59, LCDR2 comprising SEQ ID NO: 88, LCDR3 comprising SEQ ID NO: 117；
    xxi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 24, LCDR1 comprising SEQ ID NO: 60, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
    xxii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 25, LCDR1 comprising SEQ ID NO: 61, LCDR2 comprising SEQ ID NO: 89, LCDR3 comprising SEQ ID NO: 118；
    xxiii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119；
    xxiv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 27, LCDR1 comprising SEQ ID NO: 63, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 111；
    xxv. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 26, LCDR1 comprising SEQ ID NO: 62, LCDR2 comprising SEQ ID NO: 90, LCDR3 comprising SEQ ID NO: 119；
    xxvi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 111；
    xxvii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 28, LCDR1 comprising SEQ ID NO: 65, LCDR2 comprising SEQ ID NO: 91, LCDR3 comprising SEQ ID NO: 120；
    xxviii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 29, LCDR1 comprising SEQ ID NO: 66, LCDR2 comprising SEQ ID NO: 92, LCDR3 comprising SEQ ID NO: 121；
    xxix. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 30, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 93, LCDR3 comprising SEQ ID NO: 122；
    xxx. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 67, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 123；
    xxxi. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 32, LCDR1 comprising SEQ ID NO: 48, LCDR2 comprising SEQ ID NO: 82, LCDR3 comprising SEQ ID NO: 124；
    xxxii. HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 3, HCDR3 comprising SEQ ID NO: 33, LCDR1 comprising SEQ ID NO: 68, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 125；
    xxxiii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 34, LCDR1 comprising SEQ ID NO: 69, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 126；
    xxxiv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 35, LCDR1 comprising SEQ ID NO: 70, LCDR2 comprising SEQ ID NO: 94, LCDR3 comprising SEQ ID NO: 127；
    xxxv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 85, LCDR3 comprising SEQ ID NO: 128；
    xxxvi. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 36, LCDR1 comprising SEQ ID NO: 71, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 129；
    xxxvii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 115, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 130；
    xxxviii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 34, LCDR1 comprising SEQ ID NO: 70, LCDR2 comprising SEQ ID NO: 96, LCDR3 comprising SEQ ID NO: 131；
    xxxix. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 37, LCDR1 comprising SEQ ID NO: 72, LCDR2 comprising SEQ ID NO: 97, LCDR3 comprising SEQ ID NO: 132；
    xl. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 38, LCDR1 comprising SEQ ID NO: 72, LCDR2 comprising SEQ ID NO: 97, LCDR3 comprising SEQ ID NO: 132；
    xli. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 39, LCDR1 comprising SEQ ID NO: 73, LCDR2 comprising SEQ ID NO: 86, LCDR3 comprising SEQ ID NO: 133；
    xlii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 40, LCDR1 comprising SEQ ID NO: 74, LCDR2 comprising SEQ ID NO: 99, LCDR3 comprising SEQ ID NO: 134；
    xliii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 41, LCDR1 comprising SEQ ID NO: 75, LCDR2 comprising SEQ ID NO: 93, LCDR3 comprising SEQ ID NO: 135；
    xliv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 31, LCDR1 comprising SEQ ID NO: 76, LCDR2 comprising SEQ ID NO: 100, LCDR3 comprising SEQ ID NO: 130；
    xlv. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 42, LCDR1 comprising SEQ ID NO: 76, LCDR2 comprising SEQ ID NO: 100, LCDR3 comprising SEQ ID NO: 136；
    xlvi. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 43, LCDR1 comprising SEQ ID NO: 77, LCDR2 comprising SEQ ID NO: 101, LCDR3 comprising SEQ ID NO: 137；
    xlvii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 35, LCDR1 comprising SEQ ID NO: 78, LCDR2 comprising SEQ ID NO: 95, LCDR3 comprising SEQ ID NO: 138；
    xlviii. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 44, LCDR1 comprising SEQ ID NO: 79, LCDR2 comprising SEQ ID NO: 102, LCDR3 comprising SEQ ID NO: 122； or
    xlix. HCDR1 comprising SEQ ID NO: 2, HCDR2 comprising SEQ ID NO: 4, HCDR3 comprising SEQ ID NO: 45, LCDR1 comprising SEQ ID NO: 80, LCDR2 comprising SEQ ID NO: 103, LCDR3 comprising SEQ ID NO: 139.
  12. The monoclonal antibody or antigen binding fragment thereof, of claims 9-11, comprising a heavy chain variable domain (VH) having an amino acid sequence selected from the group consisting of: the amino acid sequences of: SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID  NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 188, and optionally comprising a light chain variable domain (VL) having an amino acid sequence selected from the group consisting of: SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, or SEQ ID NO: 237.
  13. The monoclonal antibody or antigen binding fragment thereof, of claim 12, comprising a combination of a heavy chain variable domain (VH) and light chain variable domain (VL) , wherein the combination is selected from the group consisting of:
    i. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 140 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 189；
    ii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 141 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 190；
    iii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 142 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 191；
    iv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 143 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 192；
    v. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 144 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 193；
    vi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 194；
    vii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 195；
    viii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 147 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 196；
    ix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 148 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 197；
    x. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 149 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 198；
    xi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 199；
    xii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 151 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 200；
    xiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 201；
    xiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 153 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 202；
    xv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 203；
    xvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 204；
    xvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 205；
    xviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 206；
    xix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 207；
    xx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 208；
    xxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 209；
    xxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 210；
    xxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 211；
    xxiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 212；
    xxv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 213；
    xxvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 165 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 214；
    xxvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 215；
    xxviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 167 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 216；
    xxix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 217；
    xxx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 218；
    xxxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 219；
    xxxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 220；
    xxxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 221；
    xxxiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 173 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 222；
    xxxv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 174 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 223；
    xxxvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 175 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 224；
    xxxvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 176 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 225；
    xxxviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 226；
    xxxix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 227；
    xl. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 179 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 228；
    xli. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 180 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 229；
    xlii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 181 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 230；
    xliii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 182 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 231；
    xliv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 183 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 232；
    xlv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 184 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 233；
    xlvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 185 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 234；
    xlvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 186 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 235；
    xlviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 236； or
    xlix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 237.
  14. The monoclonal antibody or antigen binding fragment thereof, of any one of claims 1-13, which displays one or more effector functions selected from antibody-dependent cellular cytotoxicity (ADCC) , complement-dependent cytotoxicity (CDC) , antibody-dependent cellular phagocytosis (ADCP) , and C1q binding against LAG-3 expressing cancer cells.
  15. A pharmaceutical composition, comprising said monoclonal antibody or antigen binding fragment thereof, of any one of claims 1-14, and a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.
  16. The monoclonal antibody or antigen binding fragment thereof, of claim 15, for use in human therapy.
  17. The monoclonal antibody or antigen binding fragment thereof, of claim 15, for use in preventing or treating Parkinson’s disease.
  18. The monoclonal antibody or antigen binding fragment thereof, of claim 16, for use in reducing, preventing, and/or treating an autoimmune or inflammatory disease.
  19. The monoclonal antibody, or antigen binding fragment thereof, for use according to claim 18, wherein said autoimmune or inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn’s disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Grave's disease, Hashimoto's thyroiditis, Addison’s disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis, uveitis, atherosclerosis and ankylosing spondylitis.
  20. The monoclonal antibody or antigen binding fragment thereof, for use according to claim 16, in preventing or treating cancer in a human patient.
  21. The monoclonal antibody or antigen binding fragment thereof, of claim 20, wherein said cancer is selected from the group consisting of a leukemia, a lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer) , rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma a of the lung) , bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer) , testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma.
  22. The monoclonal antibody or antigen binding fragment thereof, of claim 21, wherein said leukemia is selected from the group consisting of systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL) , T cell –ALL, acute myeloid leukemia (AML) , myelogenous leukemia, chronic lymphocytic leukemia (CLL) , multiple myeloma (MM) , chronic myeloid leukemia (CML) , myeloproliferative disorder /neoplasm, myelodysplastic syndrome, monocytic cell leukemia, and plasma cell leukemia； wherein said lymphoma is selected from the group consisting of histiocytic lymphoma and T cell lymphoma, B cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as low grade/follicular non-Hodgkin's lymphoma (NHL) , cell lymphoma (FCC) , mantle cell lymphoma (MCL) , diffuse large cell lymphoma (DLCL) , small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, and Waldenstrom's Macroglobulinemia； and wherein said sarcoma is selected from the group consisting of osteosarcoma, Ewing’s sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chrondrosarcoma.
  23. The monoclonal antibody or antigen-binding fragment thereof, for use according to any of claims 1-16, for the manufacture of a medicament to prevent, reduce, and/or treat an autoimmune or inflammatory disease in a human patient.
  24. The monoclonal antibody or antigen-binding fragment thereof, for use according to any of claims 1-16, for the manufacture of a medicament to prevent, reduce and/or treat a susceptible cancer.
  25. The antibody of any of claims 1-16, wherein the antibody is a bispecific antibody that specifically binds to LAG-3 and at least a second antigen.
  26. The antibody of claim 25, wherein the second antigen is selected from PD-1, PD-L1, CTLA-4, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS,  ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, CD47 and CD73
  27. The antibody of claim 26, wherein the bispecific antibody is conjugated to a therapeutic agent to form an immunoconjugate.
  28. A method of stimulating an antigen-specific T cell response comprising contacting said T cell with the antibody of any one of claims 1-16, such that an antigen-specific T cell response is stimulated.
  29. A method of stimulating an immune response in a subject comprising administering the antibody of any one of claims 1-16 to a subject such that an immune response in the subject is stimulated.
  30. The method of claim 29, wherein the subject is a tumor-bearing subject and an immune response against the tumor is stimulated.
  31. The method of claim 29, wherein the subject is a virus-bearing subject and an immune response against the virus is stimulated.
  32. A method for inhibiting growth of tumor cells in a subject comprising administering to the subject the antibody of any one of claims 1-16 such that growth of the tumor is inhibited in the subject.
  33. A method for treating viral infection in a subject comprising administering to the subject the antibody of any one of claims 1-16 such that the viral infection is treated in the subject.
  34. A method of stimulating an immune response in a subject comprising administering to the subject an anti-LAG-3 antibody and at least one additional immunostimulatory antibody such that an immune response in the subject is stimulated.
  35. The method of claim 34, wherein the at least one additional immunostimulatory  antibody is an anti-PD-1 antibody.
  36. The method of claim 34, wherein the at least one additional immunostimulatory antibody is an anti-PD-L1 antibody.
  37. The method of claim 34, wherein the at least one additional immunostimulatory antibody is an anti-CTLA-4 antibody.
  38. A method of treating an autoimmune or inflammatory disease in a human patient comprising administration of a monoclonal antibody or antigen-binding fragment thereof, of any one of claims 1-16.
  39. A method of treating cancer in a human patient comprising administration of a monoclonal antibody or antigen-binding fragment thereof, of any one of claims 1-16.
PCT/CN2017/074365 2017-02-22 2017-02-22 Anti-lymphocyte activation gene-3 (lag-3) antibodies and uses thereof WO2018152687A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
PCT/CN2017/074365 WO2018152687A1 (en) 2017-02-22 2017-02-22 Anti-lymphocyte activation gene-3 (lag-3) antibodies and uses thereof
US16/069,487 US10577421B2 (en) 2017-02-22 2018-02-22 Anti-LAG-3 antibodies and uses thereof
SG11201907561PA SG11201907561PA (en) 2017-02-22 2018-02-22 Anti-lag-3 antibodies and uses thereof
KR1020197014275A KR102236259B1 (en) 2017-02-22 2018-02-22 Anti-lag-3 antibodies and uses thereof
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